The effects of two short-chain perfluoroalkyl carboxylic acids (PFCAs) on northern leopard frog (Rana pipiens) tadpole development

Short-chain perfluoroalkyl carboxylic acids (PFCAs) have been detected in the environment globally. The presence and persistence of these compounds in the environment may lead to chronic wildlife exposure. We used northern leopard frog (Rana pipiens) tadpoles to investigate the chronic toxicity and the bioconcentration of two short-chain PFCAs, perfluorobutanoic acid (PFBA) and perfluorohexanoic acid (PFHxA). We exposed Gosner stage 25 tadpoles to PFBA and PFHxA (as individual chemicals) at nominal concentrations of 0.1, 1, 10, 100, and 1000 µg/L for 43–46 days. Tadpoles exposed to 0.1 to 100 µg/L of PFBA and PFHxA had significantly higher mean snout-to-vent lengths, mean masses, and scaled mass indexes than control tadpoles. These results indicate that exposure to short-chain PFCAs influences tadpole growth. Further investigation into the mechanism(s) causing the observed changes in tadpole growth is warranted. We observed a significantly higher proportion of males in the PFBA 1 µg/L treatment group, however further histological analyses are required to confirm visual sex identification before making concrete conclusions on the effects of PFCAs on amphibian sex ratios. PFBA concentrations in tissues were higher than PFHxA concentrations; a pattern that contrasts with previously published studies using fish, suggesting potential differences between taxa in PFBA and PFHxA bioconcentration. Bioconcentration factors were <10 L/kg wet weight, indicating low bioconcentration potential in tadpoles. Our results suggest that PFBA and PFHxA may have effects at environmentally-relevant concentrations (0.1–10 µg/L) and further investigation is required before these compounds can be deemed a “safe” alternative to their long-chain counterparts.


Contents
. General linear mixed model comparisons and Akaike Information Criterion (AIC) values for each predictor variable (fixed effects) combination.Predictor variables included exposure/control treatments (Treatment), snout-to-vent length (SVL), and Gosner stage (Stage) of northern leopard frog (Rana pipiens) tadpoles exposed to PFCAs in a chronic study.We included tank as a random effect in models to account for the non-independence of tadpoles in each tank.The best-fit AIC values are denoted by an asterisk (*).S5.Measured concentrations of perfluorobutanoic acid (PFBA) and perfluorohexanoic acid (PFHxA) and percent nominal concentrations detected in treatment solutions throughout the chronic experiment with Rana pipiens tadpoles.Analysis conducted on pooled samples by treatment across replicates.Method detection limits are presented when no analytical peak was detected.The method detection limit is the instrumental detection limit multiplied by the dilution factor.Table S4.Average water quality parameters (temperature ( o C), dissolved oxygen (% and mg/L), pH, conductivity (μS/cm), ammonia (mg/L), nitrate (mg/L), nitrite (mg/L)), and water hardness (mg/L)), by treatment taken throughout the chronic PFCA exposure study with Rana pipiens tadpoles.Data are reported as mean ± standard deviation.

Fig. S4
Fig. S4 Scatterplot of Rana pipiens tadpole mass (g) by Gosner stage of development following chronic exposure to PFBA.Red line shows first order polynomial relationship.Blue line indicates second order polynomial relationship (Stage^2).Green line indicates a third order polynomial relationship (Stage^3). .

Fig. S5
Fig. S5 Scatterplot of Rana pipiens tadpole mass (g) by Gosner stage of development following chronic exposure to PFHxA.Red line shows first order polynomial relationship.Blue line indicates second order polynomial relationship (Stage^2).Green line indicates a third order polynomial relationship (Stage^3). .

Fig. S1
Fig. S1 Photographs of A) female and B) male gonads from Rana pipiens tadpoles sampled from the control group of the chronic exposure study with PFCAs.Both tadpoles are Gosner stage 40.Bar represents 0.05 mm.O; Ovary T; Testis, K; Kidney.

Fig. S2
Fig. S2 Scatterplot of Rana pipiens tadpole snout-to-vent length (SVL) (mm) by Gosner stage of development following chronic exposure to PFBA.Red line shows first order polynomial relationship.Blue line indicates second order polynomial relationship (Stage^2).Green line indicates a third order polynomial relationship (Stage^3).

Fig. S3
Fig. S3 Scatterplot of Rana pipiens tadpole snout-to-vent length (SVL) (mm) by Gosner stage of development following chronic exposure to PFHxA.Red line shows first order polynomial relationship.Blue line indicates second order polynomial relationship (Stage^2).Green line indicates a third order polynomial relationship (Stage^3).

Fig. S4
Fig. S4 Scatterplot of Rana pipiens tadpole mass (g) by Gosner stage of development following chronic exposure to PFBA.Red line shows first order polynomial relationship.Blue line indicates second order polynomial relationship (Stage^2).Green line indicates a third order polynomial relationship (Stage^3).

Fig. S5
Fig. S5 Scatterplot of Rana pipiens tadpole mass (g) by Gosner stage of development following chronic exposure to PFHxA.Red line shows first order polynomial relationship.Blue line indicates second order polynomial relationship (Stage^2).Green line indicates a third order polynomial relationship (Stage^3).

Fig. S6
Fig. S6 Scatterplot of Rana pipiens tadpole scaled mass index (SMI) by Gosner stage of development following chronic exposure to PFBA.Red line shows first order polynomial relationship.Blue line indicates second order polynomial relationship (Stage^2).Green line indicates a third order polynomial relationship (Stage^3).

Fig. S7
Fig. S7 Scatterplot of Rana pipiens tadpole scaled mass index (SMI) by Gosner stage of development following chronic exposure to PFHxA.Red line shows first order polynomial relationship.Blue line indicates second order polynomial relationship (Stage^2).Green line indicates a third order polynomial relationship (Stage^3).

Table S1 .
Results from ANOVAs comparing mean water quality measures among treatment groups in the chronic PFCAs exposure study with Rana pipiens tadpoles.

Table S2 .
Results from the Kruskal-Wallis analyses median water quality measures among treatment groups in the chronic PFCAs exposure study with Rana pipiens tadpoles.

Table S3 .
General linear mixed model comparisons and Akaike Information Criterion (AIC) values for each predictor variable (fixed effects) combination.Predictor variables included exposure/control treatments (Treatment), snout-to-vent length (SVL), and Gosner stage (Stage) of northern leopard frog (Rana pipiens) tadpoles exposed to PFCAs in a chronic study.We included tank as a random effect in models to account for the non-independence of tadpoles in each tank.The best-fit AIC values are denoted by an asterisk (*).

Table S5 .
Measured concentrations of perfluorobutanoic acid (PFBA) and perfluorohexanoic acid (PFHxA) and percent nominal concentrations detected in treatment solutions throughout the chronic experiment with Rana pipiens tadpoles.Analysis conducted on pooled samples by treatment across replicates.Method detection limits are presented when no analytical peak was detected.The method detection limit is the instrumental detection limit multiplied by the dilution factor.Hyphen (-) indicates value not quantified.

Table S7 .
Results from logistic regressions examining the probability of tadpoles being male between exposure treatments and the control (intercept) in the chronic exposure study of Rana pipiens tadpoles exposed to PFBA and PFHxA.Significant results are in bold.

Table S8 .
Bioconcentration factors (BCF) calculated using average measured exposure concentrations and measured wet weight (w.w.) whole-body and liver concentrations from the 1000 µg/L PFBA and the 1000 µg/L PFHxA treatments in the chronic exposure study of Rana pipiens tadpoles.Data are shown for 3 individual tadpoles and resulting average ± SD.