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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Jamuary 2017 to 07 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
additional loading rate WAFs with no impact on results or integrity of the study (see below)
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
yes
Remarks:
additional loading rate WAFs with no impact on results or integrity of the study (see below)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Remarks:
Total Organic Carbon (TOC) analysis
Details on sampling:
TOTAL ORGANIC CARBON ANALYSIS
- Water samples were taken from the control and each test group at 0 hours (fresh media) and 48 hours (aged media) for analysis on the day of sampling
- Duplicate samples were also taken and stored frozen for further analysis, if necessary.
Vehicle:
no
Details on test solutions:
TEST WATER
- Reconstituted water (Elendt M7 Medium) used for the range-finding test is defined in Annex 2 (attached).
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST SYSTEM
- The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
- Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium (see Annex 2, attached) in a temperature controlled room maintaining the water temperature at 18 to 22 °C.
- The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20-minute dawn and dusk transition periods.
- Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin flake food suspension.
- Culture conditions ensured that reproduction was by parthenogenesis.
- Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.
- The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
Not applicable
Hardness:
Not reported
Test temperature:
21 to 22 °C (see Table 3, attached)
pH:
7.4 to 7.6 for loading rate WAFs in the definitive test (see Table 3, attached)
Dissolved oxygen:
8.5 to 9.1 mf O2/L for loading rate WAFs in the definitive test (see Table 3, attached)
Salinity:
Not applicable
Conductivity:
Not reported
Nominal and measured concentrations:
Nominal loading rates of 1.25, 2.5, 6.25, 12.5, 25, 50 and 100 mg/L in the definitive test
Details on test conditions:
VALIDATION OF MIXING PERIOD
- Preliminary work (see Annex 4, attached) was carried out to determine whether stirring for a prolonged period produced significantly higher levels of total organic carbon, as an indicator of soluble organic substances, in the WAF.

RANGE-FINDING TEST
- The test concentrations to be used in the initial experiment were determined by a preliminary 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 (5.0, 50 and 500 mg) were each separately dispensed onto a glass slide suspended within 5 Lof 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 using a stirring rate such that a vortex was formed to give a dimple at the water surface.
- Stirring was stopped after 95 hours and the mixtures allowed to stand for one hour. Visual observations made on the WAFs suggested to remove the WAFs by filtering through a glass wool plug (2-4 cm in length) as a precaution.
- 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 1.0, 10 and 100 mg/L loading rate WAFs.
- Microscopic observations of the WAFs were performed after filtering and showed there to be no undissolved test item present.
- In the range-finding test, five daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared, and maintained in a temperature controlled room maintaining the water temperature at 21 °C to 22 °C with a maximum deviation of ± 1 °C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation. After 24 and 48 hours, the number of immobilised daphnids were recorded.
- The control group was maintained under identical conditions, but not exposed to the test item.
- A sample of each test concentration was taken for Total Organic Carbon (TOC) analysis at 0 and 48 hours. All samples were stored frozen prior to analysis.

INITIAL EXPERIMENT
- Based on the results of the range-finding test, the following loading rates were assigned to the initial experiment: 0.78, 1.56, 3.13, 6.25 and 12.5 mg/L.
- A nominal amount of test item (37.5 mg) was dispensed onto a glass slide suspended within 3 L of test water to give the 12.5 mg/L loading rate.
- 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.
- Stirring was stopped after 95 hours and the mixture allowed to stand for one hour. Observations made on the WAF indicated that a significant amount of dispersed test item was present in the water column; hence, it was considered justifiable to remove the WAF 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 12.5 mg/L loading rate WAF.
- Microscopic observations of the WAF were performed after filtering and showed no test item present.
- A series of dilutions made from the 12.5 mg/L loading rate WAF to give the further loading rates of 0.78, 1.56, 3.13 and 6.25 mg/L. Each prepared loading rate was inverted several times to ensure adequate mixing and homogeneity.
- As in the range-finding test, 150 mL glass beakers containing approximately 100 mL of test preparation were used. At the start of the test five daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room maintaining the water temperature at 18 °C to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light (between 200 and 1200 Lux) and 8 hours darkness with 20-minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.
- The control group was maintained under identical conditions, but not exposed to the test item.
- The test preparations were not renewed during the exposure period.
- There was no immobilisation observed in the initial experiment, and the test series was adjusted accordingly for the definitive test.

DEFINITIVE TEST
- Based on the results of the range-finding test and initial experiment, the following loading rates were assigned to the definitive test: 1.25, 2.5, 6.25, 12.5, 25, 50 and 100 mg/L.
- Nominal amounts of test item (37.5, 75, 150 and 300 mg) were each separately dispensed onto a glass slide suspended within 3 L of test water to give the 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.
- Stirring was stopped after 95 hours and the mixtures allowed to stand for one hour.
- Microscopic observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column; 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 12.5, 25, 50 and 100 mg/L loading rate WAFs.
- Microscopic observations of the WAFs were performed after filtering and showed no undissolved test item to be present.
- Dilutions were made from the 12.5, 25 and 50 mg/L loading rate WAF to give the further loading rates of 1.25, 2.5 and 6.25 mg/L, respectively.
- Each prepared loading rate was inverted several times to ensure adequate mixing and homogeneity.

EXPOSURE CONDITIONS
- As in the range-finding 150 mL glass jars containing approximately 100 mL of test preparation were used.
- At the start of the test 5 daphnids were placed into test preparations in each test and control vessel at random.
- Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ± 1 °C and a photoperiod of 16 hours light (between 200 and
1200 lux) and 8 hours darkness with 20-minute dawn and dusk transition periods.
- The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.
- The control group was maintained under identical conditions but not exposed to the test item.
- The test preparations were not renewed during the exposure period.

TEST ORGANISM OBSERVATIONS
- Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure.
- The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

WATER QUALITY CRITERIA
- Water temperature was recorded daily throughout the test using a Hanna Instruments H93510 digital thermometer.
- Dissolved oxygen concentrations and pH were recorded at the start and termination of the test using a Hach Flexi handheld meter.
- Light intensity during the light period was measured using an ATP Instruments Lux meter.
- The appearance of the test media was recorded daily.

VALIDATION CRITERIA
- Results of the test are considered valid if the following performance criteria are met:
(a) No more than 10 % of the control daphnids show immobilisation or other signs of disease or stress (e.g. discoloration or unusual behaviour such as trapping at the surface water).
(b) The dissolved oxygen concentration at the end of the test is ≥ 3 mg/L in the control and test vessels.

MAJOR COMPUTERISED SYSTEMS
- Building management: Delta Control System
- TOC measurement: Shimadzu
- Statistical analysis: ToxRat Professional
Reference substance (positive control):
yes
Remarks:
potassium dichromate (investigation conducted between 20 october 2016 and 22 October 2016 - see Annex 3, attached)
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
19 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits 16 to 24 mg/L
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
6.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOELR
Effect conc.:
12.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
mobility
Details on results:
VALIDATION OF MIXING PERIOD
- Preliminary investigation (see Annex 4, attached) indicated that increasing the stirring period significantly increased the amount of carbon in the WAF between the 24-hour and 96-hour preparation periods.
- Preparation of the WAF therefore involved a 95-hour stirring period followed by a 1-hour standing period.

RANGE FINDING TEST
- Cumulative immobilisation data and other observations from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1 (attached).
- No immobilisation was observed at 1.0 mg/L loading rate WAF; however, immobilisation was observed at 10 and 100 mg/L loading rate WAF.
- Based on this information loading rates of 0.78, 1.56, 3.13, 6.25 and 12.5 mg/L were selected for the initial experiment.
- Total Organic Carbon (TOC) analysis of the 1.0, 10 and 100 mg/L test preparations at 0 hours (see Annex 5, attached) showed measured concentrations to range from 1.70 to 3.71 mg C/L. There was not a significant difference in the measured concentrations of carbon at 48 hours, which ranged from 1.50 to 4.22 mg C/L.

INITIAL EXPERIMENT
- No immobilization was observed in the 0.78, 1.56, 3.13, 6.25 and 12.5 mg/L loading rate WAFs after 48 hours of exposure.
- Based on the results of the range-finding test and initial experiment, a definitive test was conducted with the loading rates of 1.25, 2.5, 6.25, 12.5, 25, 50 and 100 mg/L.

DEFINITIVE TEST
- Total Organic Carbon (TOC) analysis of the test preparations at 0 hours (see Annex 5, attached) showed measured concentrations to range from less than the limit of quantification (LOQ), determined to be 1.0 mg C/L, to 5.6 mg C/L. There was no significant difference in the measured concentrations of carbon at 48 hours.
- The dissolved test item may have been one or several components of the test item. Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal test concentrations only.

IMOBILISATION DATA
- Cumulative immobilisation data and other observations from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2 (attached).
- The relationship between percentage immobilization and concentration at 48 hours is given in Figure 1 (attached).
- Analysis of the immobilisation data by Probit analysis using Linear Maximum-Likelihood regression at 24 hours and 48 hours, based on the nominal loading rates, gave an EL50 (24h) value of 27 mg/L (95 % confidence limits not determined) and an EL50 (48h) value of 19 mg/L (95 % confidence limits 16 to 24 mg/L).
- The No Observed Effect Loading Rates (NOELR) after 24 and 48 hours exposure were 25 and 6.25 mg/L, respectively.
- The Lowest Observed Effect Loading Rates (LOELR) after 24 and 48 hours exposure were 50 and 12.5 mg/L, respectively.
- The slopes and their standard errors of the response curves at 24 and 48 hours were 17 (SE = 0.12) and 3.8 (SE = 0.047), respectively.

SUB-LETHAL EFFECTS
- A sub-lethal effect of exposure was observed in the 12.5 and 25 mg/L loading rate WAFs.
- The response was reduced mobility (see Table 2, attached).

VALIDATION CRITERIA
- The test was considered to be valid given that none of the control daphnids showed immobilisation or other signs of disease or stress and that the oxygen concentration at the end of the test was ≥ 3 mg/L in the control and test vessels.

WATER QUALITY CRITERIA
- The results of the water quality measurements are given in Table 3 (attached).
- Temperature was maintained at 21 to 22 °C throughout the test.
- There were no treatment related differences for oxygen concentration or pH.
- Throughout the test the light intensity was observed to be in the range 572 to 586 lux.

OBSERVATIONS ON TEST ITEM SOLUBILITY
- At the start of stirring, the 12.5 to 100 mg/L loading rate WAFs were observed to have formed a clear, colourless water column with test item adhered to a glass slide suspended mid-depth in the media.
- At the end of stirring, the 50 mg/L loading rate WAF was observed to be a clear, colourless water column with test item adhered to a glass slide, sitting on the bottom, and test item dispersed throughout the water column.
- All other WAFs were observed to be clear, colourless water columns with test item adhered to a glass slide suspended mid-depth, and test item dispersed throughout the water column.
- Following a 1-hour standing period, the WAFs were observed to be a clear, colourless water column with test item dispersed throughout and settled on the bottom.
- At the start and throughout the test, all control and test solutions were observed to be clear, colourless solutions.
Results with reference substance (positive control):
- The results from the positive control with potassium dichromate were within the normal range for this reference item (see Annex 3, attached).
Reported statistics and error estimates:
STATISTICAL ANALYSIS
- The EL50 values and associated confidence limits at 24 and 48 hours, and the slope of the response curve and its standard error, were calculated by Probit analysis using Linear Maximum-Likelihood regression. The Lowest Observed Effect Loading Rate and the No Observed Effect Loading Rate at 24 and 48 hours were calculated using the Fisher’s Exact Binomial Test with Bonferroni correction.
- All results were calculated using the ToxRat Professional computer software package (ToxRat).
Validity criteria fulfilled:
yes
Conclusions:
Exposure of Daphnia magna to the test item gave an EL50 (48h) value of 19 mg/L loading rate WAF. The No Observed Effect Loading Rate (NOELR) was 6.25 mg/L loading rate WAF and the Lowest Observed Effect Loading Rate (LOELR) was reported as 12.5 mg/L loading rate WAF.
Executive summary:

GUIDELINE

A study was performed to assess the acute toxicity of the test item toDaphnia magna.The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202,"Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

 

METHODS

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF). Following a preliminary range finding test and initial experiment, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test item at a nominal loading rate of 1.25, 2.5, 6.25, 12.5, 25, 50 and 100 mg/L for 48 hours at a temperature of 21 to 22 °C under static test conditions. The number of immobilised daphnia were recorded after 24 and 48 hours.

 

RESULTS

Total Organic Carbon (TOC) analysis of the test preparations at 0 hours showed measured concentrations ranging from less than the limit of quantification (determined to be 1.0 mg C/L) to 5.6 mg C/L. There was no significant difference in the measured concentrations of carbon at 48 hours. 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.

 

CONCLUSION

Exposure of Daphnia magna to the test item gave an EL50 (48h) value of 19 mg/L loading rate WAF. The No Observed Effect Loading Rate (NOELR) was 6.25 mg/L loading rate WAF and the Lowest Observed Effect Loading Rate (LOELR) was reported as 12.5 mg/L loading rate WAF.

Description of key information

Exposure of Daphnia magna to the test item gave an EL50 (48h) value of 19 mg/L loading rate WAF. The No Observed Effect Loading Rate (NOELR) was 6.25 mg/L loading rate WAF and the Lowest Observed Effect Loading Rate (LOELR) was reported as 12.5 mg/L loading rate WAF (OECD 202 and EU Method C.2).

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
19 mg/L

Additional information

GUIDELINE

A study was performed to assess the acute toxicity of the test item to Daphnia magna.The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202,"Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

 

METHODS

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF). Following a preliminary range finding test and initial experiment, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test item at a nominal loading rate of 1.25, 2.5, 6.25, 12.5, 25, 50 and 100 mg/L for 48 hours at a temperature of 21 to 22 °C under static test conditions. The number of immobilised daphnia were recorded after 24 and 48 hours.

 

RESULTS

Total Organic Carbon (TOC) analysis of the test preparations at 0 hours showed measured concentrations ranging from less than the limit of quantification (determined to be 1.0 mg C/L) to 5.6 mg C/L. There was no significant difference in the measured concentrations of carbon at 48 hours. 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.

 

CONCLUSION

Exposure of Daphnia magna to the test item gave an EL50 (48h) value of 19 mg/L loading rate WAF. The No Observed Effect Loading Rate (NOELR) was 6.25 mg/L loading rate WAF and the Lowest Observed Effect Loading Rate (LOELR) was reported as 12.5 mg/L loading rate WAF.