Effectiveness of legislative tools to stop biological invasions: freshwater turtles' invasion in Europe as a study case

313 Effectiveness of legislative tools to stop biological invasions: freshwater turtles’ invasion in Europe as a study case João Rato1,2,3 , Pedro Brandão1,2,3 , Mafalda Gama1,2,3 , Filipe Banha1,2,3 , Pedro Anastácio1,2,4 1 MARE – Marine and Environmental Sciences Centre, Évora, Portugal 2 ARNET – Aquatic Research Network, Évora, Portugal 3 IIFA - Institute for Research and Advanced Training, University of Évora, Palácio do Vimioso, Largo Marquês de Marialva, 7002 - 554, Évora, Portugal 4 Department of Landscape, Environment and Planning, University of Évora, Rua Romão Ramalho, 59, Évora, Portugal Corresponding author: João Rato ([email protected]a.pt) Copyright: © João Rato et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). Research Article Abstract Invasive species pose significant threats to biodiversity, human health, and economic systems. Freshwater turtles are popular as pets, but when released or allowed to escape into the wild, they can become invasive. A prominent example is the red-eared slider Trachemys scripta, which is recognized as a globally invasive species. Legislative tools, such as the European Union Regulation 1143/2014 on the prevention and management of invasive alien species, aim to combat these threats. This framework includes the implementation of Commission Implementing Regulation 1141/2016, which banned the trade of listed invasive species, including T. scripta. This study evaluates the effectiveness of Regulation 1143/2014 on T. scripta invasion and its consequences on other freshwater turtle invasions, using data from the CITES database and the GBIF platform. While T. scripta imports into the EU ceased after 2016, the species continues to establish and expand in the wild. This suggests that the trade ban had minimal impact on already established populations, highlighting the need for additional measures to reduce and control its spread. Simultaneously, imports and records of other freshwater turtle species have risen, indicating a shift in trade dynamics. These species are increasingly being introduced, potentially exacerbating the biological invasion problem. Despite limitations inherent to CITES and GBIF data, our findings suggest that the current European legislation has not been effective in mitigating T. scripta invasions and may have unintentionally contributed to the introduction of other invasive turtles. We recommend that the European Union develop invasive species lists tailored to each biogeographic regions of Europe to enhance the effectiveness of legislation. Key words: Exotic species, management, pond turtle, regulations, terrapin Introduction The global trade of exotic animals, including both international and domestic trade, is a very lucrative business which, accounting only for the legal trade, provides revenues of millions of dollars (Lockwood et al. 2019). Globally, some of the most traded groups are birds (orders Psittaciformes and Passeriformes) and reptiles (orders Squamata and Testudines) (Bush et al. 2014). Nevertheless, domestic markets, non-traditional markets (such as websites and social media pages), and illegal markets remain undocumented or poorly documented, which makes global assessments of trade difficult (Lockwood et al. 2019). On the other hand, “legal” Academic editor: Tammy Robinson-Smythe Received: 2 December 2024 Accepted: 9 April 2025 Published: 7 October 2025 Citation: Rato J, Brandão P, Gama M, Banha F, Anastácio P (2025) Effectiveness of legislative tools to stop biological invasions: freshwater turtles’ invasion in Europe as a study case. In: Anastácio P, Ribeiro F, Chainho P (Eds) Invasions in Aquatic Systems. NeoBiota 102: 313–329. https://doi.org/10.3897/ neobiota.102.143330 NeoBiota 102: 313–329 (2025) DOI: 10.3897/neobiota.102.143330 Advancing research on alien species and biological invasions A peer-reviewed open-access journal NeoBiota 314 NeoBiota 102: 313–329 (2025), DOI: 10.3897/neobiota.102.143330 João Rato et al.: Effectiveness of legislative tools to stop biological invasions international trade is well documented. For example, the international trade of exotic species that are threatened is regulated by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). Depending on its threatened status, a species trade may only be authorised under special circumstances (Appendix I, CITES 2025), may be subjected to strict rules not to endanger the species (Appendix II, CITES 2025) or may even have special rules for specific parties (Appendix III, CITES 2025). Convention parties are obliged to report imports and exports of the species included in the CITES appendices, these are gathered in an international database (https://trade.cites.org/). However, many species are not regulated by CITES because they are not considered threatened and thus remain freely traded (Bush et al. 2014). The need to document species trade is not only related to identifying and regulating the trade of endangered species but also to identifying and preventing the introduction of exotic species (Gippet and Bertelsmeier 2021). The commercial success of a species can increase its invasive potential, as the most traded species are also the ones with the potential of being most introduced outside their native range (Gippet and Bertelsmeier 2021). Like many exotic animals kept as pets, freshwater turtles (FTs) are often released to the wild when they get bigger and older (Banha et al. 2019). As a result, FTs have become invasive worldwide and, in some cases, with great success, as the red-eared slider (Trachemys scripta THUNBERG, 1792) (European Environment Agency 2012). This species has been introduced to virtually every continent, mainly through the pet market, and has severe impacts on biodiversity and human health (European Environment Agency 2012). At the biodiversity level, T. scripta may impact native FTs, by competing for food and thermoregulation sites, displaying aggressive behaviour, and/or disease transmission (Polo-Cavia et al. 2014; Rato et al. 2024b). Due to their opportunistic feeding behaviour and their dietary ontogenic shift, T. scripta feeds on a variety of animal and plant species, which has implications for aquatic communities (Pérez-Santigosa et al. 2011; European Environment Agency 2012). Regarding human health, T. scripta is a well-known reservoir of Salmonella spp., which has caused several pet turtle-associated salmonellosis outbreaks in the USA (Basler et al. 2015). These turtles may also harbour other zoonotic disease agents, like Leptospira sp. and Chlamydia spp., among others (Mitura et al. 2017; Bonacina et al. 2021). To reduce the impacts and minimise introduction risk of these species, several actions may be taken such as environmental education, prevention, control and eradication of established populations. One of the possible actions is the creation of legislation to prevent the introduction of non-native and invasive species. Such legislation, typically including species’ blacklists, may prohibit the trade, the reproduction, and the maintenance under captivity of certain non-native and invasive species that are particularly damaging to the ecosystems, or in some cases, that might be disease vectors (Simberloff 2006; Zamora-Marín et al. 2023). However, these blacklists (1) often include some, but not all, of the already introduced species in an area/country, (2) do not prevent the spread of established species or the substitution of their trade by similar species, and (3) do not involve any financial support for their implementation (Simberloff 2006; Oficialdegui et al. 2023; Zamora-Marín et al. 2023). To complement the blacklists, some countries have created whitelists, i.e. lists of authorized species to trade, which may be associated with a risk analysis (Simberloff 2006). In the European Union, in 1996 the Council Regulation (EC) 338/97, which regulated the trade of some exotic species was 315 NeoBiota 102: 313–329 (2025), DOI: 10.3897/neobiota.102.143330 João Rato et al.: Effectiveness of legislative tools to stop biological invasions created, but only included one FT, T. scripta. However, the implementation of this regulation alone was insufficient to halt the spread of invasive species. To address this, a subsequent regulation, Regulation 1143/2014, was introduced. It includes a list of non-native species of Union Concern whose trade is prohibited by implementing regulation 1141/2016. However, this regulation initially listed only one freshwater turtle (FT). It is worth noting that the list of species of Union concern may differ from those established at the national level, i.e. the lists of species of national concern as per Article 12 of Regulation 1143/2014. For example, the Portuguese blacklist includes five non-native turtles (Macrochelys temminckii, Chelydra serpentina, Pseudemys spp., Graptemys spp. and Trachemys spp.) while the Spanish blacklist includes three non-native freshwater turtles (Chrysemys picta, Pseudemys peninsularis and Trachemys scripta) (Decreto-Lei n.o. 92/2019, 2019; Real Decreto 216/2019, 2019). The traditional field monitoring of non-native and invasive species may be extremely difficult and time-consuming, and some projects and studies are starting to incorporate data from citizen science projects (Johnson et al. 2020). Although it has some constraints, citizen science may be an effective way to gather long-term and large amounts of species distribution data (Johnson et al. 2020; Larson et al. 2020). For invasive species monitoring, it may be particularly important to record the first detection of a species, to update the distribution of non-native/invasive species and to use distribution data to predict future distribution ranges (Johnson et al. 2020). Some projects are species-specific (Johnson et al. 2020; Larson et al. 2020), which may limit the detection of different non-native species. The Global Biodiversity Information Facility (GBIF) is an international database that compiles data information from multiple data sources, including from several citizen science projects, for many of the world’s species. This open-access database may be used to monitor non-native and invasive species (Ivanova and Shashkov 2021). To evaluate the impact of legislation in preventing and mitigating the invasive species introductions associated with the pet market, we used the freshwater turtle’s invasion in Europe as a case study. Namely, we focused on the effectiveness of the 1143/2014 European Union regulation on T. scripta invasion, included in the implementing regulation 1141/2016, comparing EU with non-EU countries. We analyzed T. scripta number of imports and records in the wild for all European countries, both EU members and non-EU members. Similarly, we also gathered imports and records in the wild, for other species of FTs. We expected to find a reduction in the imports of T. scripta along with a stabilization or reduction of T. scripta records, potentially accompanied by an increase in the imports and records of other FTs, particularly in EU member states. Methods Data acquisition and datasets Importation data was acquired from the CITES database on February 2nd 2024. We chose to only use CITES listed species because this is the longest running import dataset, using data from 1998 until 2022. Other data on international trade of FTs is scarce. We started in 1998 because the mandatory report of CITES listed species for EU countries only started in June 1997 (Council Regulation (EC) 338/97 1996). Trachemys scripta is not listed as an endangered species by any of 316 NeoBiota 102: 313–329 (2025), DOI: 10.3897/neobiota.102.143330 João Rato et al.: Effectiveness of legislative tools to stop biological invasions the appendices of CITES; however, due to its invasive status, it was added to the transcription of CITES to the EU legislation, and therefore, this species’ importations are also reported and added to the CITES database. We did not incorporate any data or estimates from domestic and illegal trade of non-native freshwater turtles. From the CITES dataset, we only retrieved data from European countries and excluded any imports of dead specimens, animal parts, and eggs, leaving only live juvenile and adult turtles. All importation purposes, from pet to research, were included in the retrieved dataset. The total number of turtles imported for each purpose can be consulted in Suppl. material 1: table S1. To standardize all importation data, imports reported in kg were divided by the mean reported species weight to get the number of live specimens. We grouped all imports of other FTs in a single variable. On February 15th 2024, we retrieved from GBIF all European observation records for all the CITES listed freshwater turtle species for each year for the same period (1998–2022). We excluded data that came from fossil records, preserved specimens, material citations, and sample citations, leaving only live specimens’ observations. Like for the CITES dataset, we separated T. scripta observations and grouped the remaining FTs’ observations into a single variable, for each year and country. The two datasets were then joined by the corresponding year and country. Statistical analysis For the modeling approach, the spatio-temporal variation of import and presence in the wild data for freshwater turtles was explored using linear mixed models (LMMs). LMMs were used because traditional statistical regression methods are not adequate when variables have non-linear relationships, as is expected for most biological contexts. LMMs have also been widely used for modeling data on invasive species with a wide distribution and spatial/temporal dependence, taking into account random factors (Thiele and Markussen 2012; Costa 2014). For each of the models, the response variables were modelled using Period (before vs after the regulation 1143/2014), EU membership, Country population, Trachemys scripta imports and records and other FTs’ imports and records (i.e. fixed effects). The GBIF records were aggregated as the total number of T. scripta records per country per year. Similarly, for other FTs, the GBIF records were calculated as the total number of non-T. scripta FT records per country per year. Year, GDP, Country area and Country were treated as random factors to consider possible variations in terms of the legislation enforced. For the records models we also used the number of GBIF records of testudines species in Europe as random factor. The use of random factors allowed to account for the variability not explained by the fixed effects. For the variable Period we considered two periods: Before 2016 and After 2016. We chose 2016 because it was the year of publication of implementing regulation 1141/2016, which classified T. scripta as invasive species of Union concern and prohibited its trade in the EU. For each year, we classified the countries as EU or non-EU members according to their date of entry to the EU; this resulted in a different number of countries in each year for both countries’ groups (Suppl. material 1: table S2). To check for multicollinearity and redundancy of variables, following Fielding and Haworth (1995), a Spearman correlation analysis was conducted. Only Year and number of CITES listed species, as well as Country and Country area showed a high correlation coefficient (r > 0.9), and therefore we excluded the 317 NeoBiota 102: 313–329 (2025), DOI: 10.3897/neobiota.102.143330 João Rato et al.: Effectiveness of legislative tools to stop biological invasions number of listed CITES species and Country area from our analysis (Suppl. material 1: fig. S1). The best model was obtained by considering models with different variable combinations and selecting the best fit model using Akaike information criterion (AIC), with the most parsimonious models presenting the smallest AIC (goodness-of-fit measure) (Vrieze 2012). Post-hoc comparisons after LMM were performed to estimate pairwise differences between the categorical variables using Tukey-HSD tests including p-value corrections for multiple comparisons, summarized in Suppl. material 2. A statistical significance level of 0.05 was considered in all test procedures. Statistical analyses were performed within the R Software (Version 1.1.463) (Posit team. 2022) using the “mgcv” (Wood 2011), “ggplot2” (Wickham 2016) and “lme4” (Bates et al. 2015) packages. The limitations of using the CITES database and GBIF records to assess the effectiveness of Regulation 1143/2014, as well as their implications for other exotic freshwater turtles, are discussed in Suppl. material 3. Results The top 10 most traded species in European countries according to CITES and the top 10 species with most records on GBIF, excluding Trachemys scripta, are identified in Table 1. Five species – Graptemys pseudogeographica, Mauremys reevesii, Mauremys sinensis, Graptemys ouachitensis, and Pelodiscus sinensis – rank among the most frequently traded and recorded species. The best Trachemys scripta imports model was composed by period, EU membership and the interaction between period and EU membership, with the interaction presenting the only significant result (p-value = 0.0287, Table 2). Tukey test results showed significant differences between T. scripta imports in EU and non-EU countries before the legislation and between the period before and after the legislation for EU countries (Suppl. material 2, but also visible in Fig. 1A). Data on annual imports show that T. scripta imports start to decrease from 1998 until 2000 when they started to stabilize at low numbers and completely stopped in 2016. By opposition other FTs’ imports remained stable until 2004 when they started to increase, with a peak in 2018 for EU countries and a peak in 2021 for non-EU countries (Suppl. material 1: fig. S2), despite of the influence of T. scripta imports on the other FTs’ imports being non-significant (Table 2). The main European importers were Spain, Portugal, and Belgium (Fig. 2A). For T. scripta records the best model was the one that incorporated period and EU membership. However, none of these were significant in their own right (Table 2). T. scripta records increased since 1998 for both country groups, but with a slight stabilization in the later years for non-EU countries. This differs from T. scripta imports, which had a huge number of importations between 1998 and 2000 and stopped after 2016 (Fig. 1A, B; Suppl. material 1: fig. S2). Overall, the increase in T. scripta records was correlated with an increase in all testudines records in Europe (r2= 0.9, Suppl. material 1: figs S1, S3). When looking at the difference between the EU members and non-members, T. scripta records had increased in both countries’ groups, although the total number of records was different, being higher in EU countries (Fig. 1A, B). The countries with the largest number of records per km2 were Belgium, The Netherlands, Luxembourg, and Spain, which all belong to the EU (Fig. 2B). Overall, T. scripta records were higher after 2016, compared to the previous period, for EU countries (Table 2, Fig. 1B). 318 NeoBiota 102: 313–329 (2025), DOI: 10.3897/neobiota.102.143330 João Rato et al.: Effectiveness of legislative tools to stop biological invasions Table 1. Top 10 most imported and top 10 most recorded freshwater turtles from 1998 to 2022 for all Europe (combined EU and Non-EU countries), apart from Trachemys scripta, ranked by decreasing order. Species included in both lists are in bold. Most traded species on CITES Most recorded species on GBIF Family Species Family Species Emydidae Graptemys pseudogeographica Emydidae Graptemys pseudogeographica Geoemydidae Mauremys reevesii Emydidae Pseudemys concinna Geoemydidae Mauremys sinensis Chelydridae Chelydra serpentina Emydidae Graptemys ouachitensis Emydidae Pseudemys nelsoni Pelomedusidae Pelomedusa subrufa Emydidae Graptemys ouachitensis Trionychidae Pelodiscus sinensis Geoemydidae Mauremys sinensis Pelomedusidae Pelusios niger Emydidae Chrysemys picta Emydidae Graptemys versa Geoemydidae Mauremys reevesii Podocnemididae Podocnemis unifilis Emydidae Pseudemys rubriventris Pelomedusidae Pelusios castaneus Trionychidae Pelodiscus sinensis Table 2. Results of the selected LMM to explain variation on the imports and records of Trachemys scripta and other FTs per squared kilometre and species. All variables included in the models were significant. Model (n = 2070) Model term Fixed effects Random effects Estimate SE P-value Variance SD Trachemys scripta imports Period: Before 2016 -11.84 100.225 0.9059 – – EU: Yes 1.069 68.9202 0.9876 – – Period × EU 178.303 81.5145 0.0287 – – (1|Year) – – – 33272 5168 (1|Country) – – – 3780 182.41 Trachemys scripta records Period: Before 2016 -0.7396 0.8611 0.3903 – – EU: Yes -0.5924 1.3871 0.6693 – – (1|All records) – – – 122579.46 350.114 (1|Year) – – – 0.000e+00 0.0000 (1|Country) – – – 118.40 10.881 (1|GDP) – – – 0.000e+00 0.0000 Other FTs’ imports Period: Before 2016 3.293e+02 2.080e+03 9.001e-01 – – EU: Yes 1.088e+04 3.131e+03 5.216e-04 – – Period x EU -1.138e+04 2.641e+03 1.631e-05 – – Imports Trachemys scripta -1.615e+00 9.501e-01 8.904e-02 – – (1|Year) – – – 3412347 0.08907 (1|Country) – – – 182548370 1847.3 (1|GDP) – – – 185035 430.2 Other FTs’ records Imports other 7.735e-06 1.841e-06 2.66e-05 – – (1| All records) – – – 2.024e+02 14.22653 (1|Year) – – – 3.159e-03 0.05621 (1|Country) – – – 0.000e+00 0.00000 (1|GDP) – – – 6.304e-04 0.02511 319 NeoBiota 102: 313–329 (2025), DOI: 10.3897/neobiota.102.143330 João Rato et al.: Effectiveness of legislative tools to stop biological invasions Figure 1. Trachemys scripta annual variation of imports and records in nature, between 1998 and 2022. Red lineEU member states. Blue linenon-EU countries A total imports by year in CITES database B total records in GBIF per year. Figure 2. Trachemys scripta imports and records in Europe (both EU and Non-EU countries), combined between 1998 and 2022 A total imports per country per square kilometre B total records per country per square kilometre. Increasing number of imports (A) and (B) from lighter to darker colours. Grey colour corresponds to absent data. The best model for other FTs’ imports included Period, T. scripta imports, EU membership and the interaction between the period and EU membership. For this model only T. scripta imports and Period were non-significant (p-value = 0.0967). The import of other FTs reached its maximum in 2019 (Fig. 3A; Suppl. material 1: fig. S2). Before 2005, the imports of other FTs were very low. Like in the other models, the number of imported turtles was higher in EU countries (Table 2 and 320 NeoBiota 102: 313–329 (2025), DOI: 10.3897/neobiota.102.143330 João Rato et al.: Effectiveness of legislative tools to stop biological invasions Figure 3. Other non-Trachemys scripta freshwater turtles’ annual variation of imports and of records in nature. Red lineEU member states. Blue linenon-EU countries A total imports by year in the CITES database B total records in GBIF per year. Figure 4. Other non-Trachemys scripta freshwater turtle imports and records in Europe (both EU and Non-EU countries), combined between 1998 and 2022 A total imports per country, per square kilometre, per CITES listed species B total records per country, per square kilometre. Increasing number of imports (A) and (B) from lighter to darker colours. Grey colour corresponds to absent data. Fig. 4A). The countries with the largest number of imports were Portugal, Italy and Luxembourg (Fig. 4A). The Tukey test was significant for all interactions except for non-EU countries between, before and after 2016 (Suppl. material 2). Finally, regarding other FTs’ records, the best model only included the variable of other FTs’ imports, and it was significant (p-value = 2.66e-05, Table 2). The number of other FTs’ records was small, with a substantial increase from 2005 onwards (Fig. 3B, Suppl. material 1: fig. S4). The countries with the largest number 321 NeoBiota 102: 313–329 (2025), DOI: 10.3897/neobiota.102.143330 João Rato et al.: Effectiveness of legislative tools to stop biological invasions of records all belong to the EU, namely Portugal, Spain, The Netherlands, and Belgium (Fig. 4B). The remaining countries only have occasional records by square kilometer (Fig. 4B). Discussion From our results, we can conclude that, despite the legislation leading to a reduction in T. scripta imports, the records of this species did not decrease in the wild. On the other hand, other freshwater turtles (FT) imports increased with the legislation implementation, and these imports could at least partially explain the increase in records of other FTs in the wild. Imports of T. scripta ceased entirely with the implementing Regulation 1141/2016, which may lead to a reduction of new introductions of this species. Additionally, propagule pressure is also important at the post-establishment stages of invasion (Colautti and MacIsaac 2004), as it promotes the arrival of new propagules from the outside of the established populations. Although international trade was stopped, the same might not have happened with the in-country and illegal trade. This was also found for other taxonomic groups, for instance, in the Australian ornamental fish industry, several black-listed species continue to be traded, particularly through e-commerce (Millington et al. 2022). This might be reduced by applying strong control to the internal trade of pet species, and not focusing only on international trade (Millington et al. 2022). It is relevant to note here that the mean imports of T. scripta in non-EU countries are very low compared to EU countries, which might be related to the low GPD of non-EU countries (Evans et al. 2018). Even while T. scripta imports decreased between 1998 and 2000 and reached a relatively low level after that year, records did not follow the same trend. After 2003, T. scripta records were continuously increasing, and in 2018, there was a large increase in the number of records that was maintained high until 2022. This aligns with the general trend observed in the records of all turtle species in Europe, where a mean of 40% (ranging from 17% to 64%) of annual records are attributed to T. scripta. In our statistical analysis we included, as a random factor, the records of all turtle species in Europe to account for the increasing use of citizen science platforms over the years, and so our results already consider that effect. Despite not being statistically significant, the variable T. scripta imports follows the opposite trend of the T. scripta records. This is an indication that the imports’ reduction did not affect the records in the wild, and therefore a blacklist alone has not stopped the invasion of T. scripta. The same happened with T. scripta records after the national legislation implementation in Spain (Maceda-Veiga et al. 2019). Although legislation implementation increased the voluntary surrendering of animals to the competent authorities and the confiscation of prohibited species, the number of wild/feral individuals remained unchanged (Maceda-Veiga et al. 2019). 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NeoBiota 89: 17–44. https://doi.org/10.3897/neobiota.89.105994 Supplementary material 1 Supplementary figures S1–S4 and tables S1, S2 Authors: João Rato, Pedro Brandão, Mafalda Gama, Filipe Banha, Pedro Anastácio Data type: pdf Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/neobiota.102.143330.suppl1 Supplementary material 2 Tukey test results Authors: João Rato, Pedro Brandão, Mafalda Gama, Filipe Banha, Pedro Anastácio Data type: xlsx Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/neobiota.102.143330.suppl2 Supplementary material 3 Supplementary data note Authors: João Rato, Pedro Brandão, Mafalda Gama, Filipe Banha, Pedro Anastácio Data type: pdf Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/neobiota.102.143330.suppl3