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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 October 2017 to 29 March 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Nominal loading rates of 6.25, 12.5, 25, 50 and 100 mg/L
- Sampling method: Prepared as a Water Accommodated Fraction (WAF)
- Sample storage conditions before analysis: Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 20°C
Vehicle:
no
Details on test solutions:
Range-finding Test:
In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/L.
Nominal amounts of test item (20, 20 and 200 mg) were each separately added to the surface of 20, 2 and 2 liters of test water to give the 1.0, 10 and 100 mg/L loading rates, respectively. After the addition of the test item, the test water was stirred by magnetic stirrer at a rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75 to 100 mL discarded) to give the 1.0, 10 and 100 mg/L loading rate WAFs.

Definitive Test
Based on the results of a preliminary range-finding test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to a WAF of the test item over a range of test concentrations of 6.25, 12.5, 25, 50 and 100 mg/L for a period of 21 days. The test solutions were renewed three times per week throughout the test.
Nominal amounts of test item (12.5, 25, 50, 100 and 200 mg) were each separately added to the surface of 2 liters of test water to give the 6.25, 12.5, 25, 50 and 100 mg/L loading rates, respectively. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for1 hour. During the first and second preparations, a wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75 to 100 mL discarded) to give the 6.25, 12.5, 25, 50 and 100 mg/L loading rate WAFs. During the third renewal interval and onwards, observations made on the WAFs after stirring indicated that a significant amount of undissolved test item was dispersed throughout the water columns, and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 6.25, 12.5, 25, 50 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed that no undissolved test item remained.

No filtration was required during the range-finding test. The Day 0 freshly prepared 1.0, 10 and 100 mg/l loading rates yielded measured concentrations in the range of less than the LOQ at 1.0 and 10 mg/l to 0.052 mg/l at 100 mg/l loading rate WAF. For the definitive test the freshly prepared Day 0 and Day 5 WAFs did not require filtration. Analysis of the Day 0 at 100 mg/l loading rate WAF preparation showed a measured concentration of 0.053 mg/L was obtained, whilst a measured concentration of less than the LOQ was obtained on Day 5. The Day 9, 14 and 19 fresh preparations were all filtered through a glass wool plug. Measured concentrations of less than the LOQ to 0.016 mg/L were obtained. These results suggest that the decline in measured concentration to less than the LOQ was not attributable to the use of a glass wool plug. The filtration does not impact the composition of the test material. No adverse effects were observed at 100 mg/l loading rate WAF in either the range-finding or definitive test. For more details, please see the analytical report attached in the background document part.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Source: In house laboratory cultures
- Feeding during test: Yes.
- Food type: mixture of algal suspension and Tetramin flake food suspension.
- Frequency: Daily.


METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the yound daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
244 to 276 mg/L as CaCO3
Test temperature:
19 - 22°C
pH:
7.8 - 8.2
Dissolved oxygen:
=8.1 mg O2/L
Nominal and measured concentrations:
Nominal : 6.25, 12.5, 25, 50 and 100 mg/L
Details on test conditions:
For each concentration adult daphnids were placed in 100 mL of the test preparation in 150 mL glass vessels (one daphnid per vessel), which were then covered with a plastic lid to reduce evaporation. For each test and control group 10 replicate test vessels were prepared. The test vessels were maintained in a temperature controlled room at 18 to 22°C with a maximum deviation of +-1°C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for 21 days. The diluent water was aerated prior to use, but the test vessels were not aerated during the exposure.

The control group was maintained concurrently under identical conditions, but was not exposed to the test item.
Key result
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
- Effects on Reproduction : There was no significant mortalities observed in the parental generation (P1) and there were not significant differences (P>=0.05) in terms of the number of live young produced per adult when compared to the control following the exposure to the test item for 21 days.
- Behavioural abnormalities: There was no significant effect on the size and color of the parental daphnids at any of the loading rates tested.
- Observations on body length and weight: see more detailed results on body length in the background document part.
- Other biological observations: Two mortalities were observed at 50 mg/L loading rate WAF, one each on Days 11 and 15. Given that the observed parental mortality was not statistically significant and did not follow a concentration-response pattern, these mortalities were assigned as inadvertent and offspring from these two parental daphnids were excluded from the assessment of reproductive effects. No mortalities occurred at 6.25, 12.5, 25 and 100 mg/L loading rate WAF throughout the test.
- Mortality of control: A single adult daphnid was observed to be immobilized in the control group on Day 12, however, as no more than 10% were affected, this was considered an inadvertent mortality attributable to natural causes and as such considered not to have had an impact on the outcome of the test.
- Other adverse effects control: A single parental daphnid in the control group was observed to be pale from Day 16 to Day 20. A single parental daphnid in the 50 mg/L loading rate WAF test group was observed to be pale on Days 13 to 17 and on Day 20, and a single parental daphnid in the 100 mg/L loading rate WAF test group was observed to be pale on Day 18. Given that these were transient effects that were not loading rate dependent, these were considered to be due to natural causes and as such were not considered to be treatment related.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none. After removal of the aqueous phase by siphon or filtration, the control, 6.25, 12.5, 25, 50 and 100 mg/L loading rates were observed to be clear, colorless solutions.
Reported statistics and error estimates:
For the estimation of the No Observed Effect Loading Rate (NOEL), the numbers of live young produced per adult over the duration of the test for the control and each test group were compared using the Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm procedure. Daphnid length in the control and each test group, as determined for the surviving daphnids at test termination, was compared using one way analysis of variance incorporating Bartlett’s test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett’s multiple comparison procedure for comparing several treatments with a control (Dunnett 1955), using the SAS computer software package (SAS 1999 - 2001).
Validity criteria fulfilled:
yes
Conclusions:
Exposure of Daphnia magna to the test item for 21 days resulted in no significant mortalities at any of the loading rates employed during the test.

The No Observed Effect Loading Rate for the parental daphnia generation (P1) survival was determined to be greater than 100 mg/L loading rate.

No significant effects on reproduction were observed at the loading rates employed during the test.

The 21 day EL50 (reproduction) based on nominal loading rates was greater than 100 mg/L.

The No Observed Effect Loading Rate based on nominal loading rates was 100 mg/L.
Executive summary:

A study was performed to assess the chronic toxicity of the test item, to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines No. 211 and GLP. Due to the low aqueous solubility and complex composition of the test item, the test medium was prepared as a Water Accommodated Fraction (WAF).


The dissolved fraction of the WAF test solution may have been comprised of one or several components of the test item. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.


Chemical analysis of the fresh test preparations on Days 0, 5, 9, 14 and 19 showed measured test concentrations ranged from less than the limit of quantification (LOQ, 0.011 mg/L) of the analytical method to 0.053 mg/L. Chemical analysis of the aged test preparations on Days 2, 7, 12, 16 and 21 showed measured test concentrations ranged from less than the LOQ to 0.023 mg/L.


The 21-Day EL50 (immobilization and reproduction) value, based on nominal loading rates, was estimated to be greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate (NOEL) was considered to be 100 mg/L on the basis that at this test concentration there were no significant mortalities (immobilization) observed in the parental generation (P1) and that there were no significant differences (p>0.05) between the control and the 100 mg/L loading rate WAF test group in the numbers of live young producd per adult by Day 21. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

Description of key information

In an OECD 211 study conducted according to GLP, the long-term toxicity of the test item to the freshwater invertebrate Daphnia magna, was investigated under semi-static conditions. The 21-Day EL50 values for immobilization in the parental daphnia generation (P1) and reproduction, based on nominal loading rates, were estimated to be greater than 100 mg/L loading rate WAF. The No Observed Effect Loading rate (NOEL) was considered to be 100 mg/L on the basis that at this test concentration there were no significant mortalities (immobilization) observed in the parental generation (P1) and that there were no significant differences (p>0.05) between the control and the 100 mg/L loading rate WAF test group in the numbers of live young produced per adult by Day 21.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
NOEC
Remarks:
No Observed Effect Loading rate (NOEL)
Effect concentration:
100 mg/L

Additional information