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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:
30 September 2014 to 28 October 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
water hardness measurement omitted in 100 % v/v saturated solution on Day 16 with no impact on outcome of study (see below)
Qualifier:
according to
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
water hardness measurement omitted in 100 % v/v saturated solution on Day 16 with no impact on outcome of study (see below)
Qualifier:
according to
Guideline:
EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)
Deviations:
yes
Remarks:
water hardness measurement omitted in 100 % v/v saturated solution on Day 16 with no impact on outcome of study (see below)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
CHEMICAL ANALYSIS OF TEST LOADING RATES
- Water samples were taken from the control and each surviving test group for quantitative analysis (replicates pooled).
- Samples of fresh test preparations were taken on Days 0, 5, 9, 14 and 19.
- Samples of expired test preparations were taken on Days 2, 7, 12, 16 and 21.
- Samples were stored frozen prior to analysis.
- Duplicate samples were taken and stored frozen for further analysis if necessary.

TOTAL ORGANIC CARBON ANALYSIS
- Water samples were taken from the control and each surviving test group for quantitative analysis (replicates pooled).
- Samples of fresh test preparations were taken on Days 0, 5, 9, 14 and 19.
- Samples of expired test preparations were taken on Days 2, 7, 12, 16 and 21.
- Samples were analysed on the day or stored at approximately 4 °C prior to analysis.
- Duplicate samples were taken and stored at approximately 4 °C for further analysis if necessary.

Vehicle:
no
Details on test solutions:
TEST WATER
- Reconstituted water (Elendt M7 medium) was used to maintain the stock animals and for the definitive test (see details given in Appendix 1, attached).
Test organisms (species):
Daphnia magna
Details on test organisms:
- The test was performed 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 Appendix 1, attached) in a temperature controlled room at approximately 20 °C.
- The lighting cycle was controlled to give 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.
- Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetram in 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 prod uced overnight were less than 24 hours old. These young were removed from the cultures and used for
testing.
- The diet and diluent water were considered not to contain any contaminant that would affect the integrityor outcome of the study.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
Not applicable
Hardness:
238 to 282 mg/L as CaCO3 (see Table 11, attached)
Test temperature:
20 to 21 °C (see Table 10, attached)
pH:
7.3 to 8.8 (see Table 10, attached)
Dissolved oxygen:
8.1 to 9.4 mg O2/L (see Table 10, attached)
Salinity:
Not applicable
Conductivity:
Not reported
Nominal and measured concentrations:
DEFINITIVE TEST
- Nominal concentrations of 0.31, 0.63, 1.25, 2.5 and 5.0 % v/v saturated solution
Details on test conditions:
PRELIMINARY MEDIA PREPARATION TRIAL
- Preliminary solubility work indicated that the test item was practically insoluble in water using traditional methods of preparation such as ultrasonication and high shear mixing.
- Based on this information, the test item was categorised as a ‘difficult substance’ in accordance with the OECD guidance document on aquatic toxicity testing of difficult substances and mixtures (OECD; 2000).
- A media preparation trial was therefore conducted to determine the solubility of the test material under test conditions (see Appendix 2 attached).

DEFINITIVE TEST
- Based on the results of an acute toxicity test, Daphnia magna were exposed to test item (10 replicates of a single daphnid per group) for 21 days over the concentration range 0.31, 0.63, 1.25, 2.5 and 5.0 % v/v saturated solution.
- Test solutions were renewed three times per week throughout the test.
- A nominal amount of test item (400 mg) was added to the surface of 4 L of test water to give the 100 mg/L loading rate.
- After addition of the test item, the test water was stirred for 23 hours at a rate such that a dimple was formed on the surface of the water.
- Stirring was stopped after 23 hours and the media was allowed to stand for one 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 then inserted into the glass tube and pushed through the Nescofilm seal.
- Microscopic inspection of the aqueous pahase showed no micro-dispersions or undissolved test item to be present.
- The aqueous phase was removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 100 % v/v saturated solution.
- Diltutions were performed from the saturated solution to give the test series.
- Total Organic Carbon (TOC) and boron concentrations in the test preparations were verified on Days 0, 5, 9, 14 and 19 (fresh media) and Days 2, 7, 12, 16 and 21 (old media) (see Appendix 3 and Appendix 4, attached).
- For each concentration, a single daphnid was placed in 100 mL of the test preparation in 150 mL glass vessels, which were then covered with a plastic lid to reduce evaporation.
- For each test and control group, ten replicate test vessels were prepared.
- The test vessels were maintained in a temperature controlled room at approximately 21 °C for 21 days with a photoperiod of 16 hours light (510 to 677 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods.
- Each vessel was randomly assigned to a position in the laboratory.
- Test vessels were not aerated.
- The diluent water was only aerated prior to use.
- The control group was maintained under identical conditions but not exposed to the test item.
- Test preparations were renewed three times per week on Days 0, 2, 5, 7, 9, 12, 14, 16 and 19. The adult Daphnia were transferred to fresh media by wide-bore pipette before the contents of each vessel was passed through a fine mesh. Young daphnids (live and dead) and any unhatched eggs were collected on the mesh and counted before being discarded.
- Each daphnid received approximately 5 to 15 µL of an algal suspension (Desmodesmus subspicatus) and approximately 10 to 70 µL of Tetramin flake food suspension daily. Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day and was dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.

TEST ORGANISM OBSERVATIONS
- On a daily basis, the numbers of live and dead from the parental (P1) generation, the numbers of live and dead filial (F1) Daphnia, and the number of discarded unhatched eggs were counted. An assessment was also made of the general condition and size of the parental Daphnia as compared with the controls.
- The number of Daphnia with eggs or young in the brood pouch was determined daily.
- Young daphnids were considered to be dead if no sign of movement was apparent during microscopic examination.
- Immobile adult Daphnia, which were unable to swim for approximately 15 seconds after gentle agitation, were considered to be dead.
- An immobilisation criteria for the young daphnids was considered to be inappropriate due to the large numbers of offspring produced in the flasks.
- At the end of the test, the length of each surviving parent animal was determined.

WATER QUALITY CRITERIA
- Dissolved oxygen concentrations, pH and temperature were recorded before and after each test media renewal.
- The pH and dissolved oxygen concentration were measured using a Hach HQ30d Flexi handheld meter.
- Temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
- Measurements were made on one replicate for each test concentration.
- The temperature was also measured every hour in one replicate of the control using a Testo temperature logger.
- Water hardness of the control and the highest surviving test concentration in the fresh and old media was measured once per week. However, on Day 16, no sample was analysed for the old media in the highest surviving test concentration. This was a deviation to the study plan but was considered to have had no impact on the outcome or integrity of the study because the hardness was consistent on all other occasions.

VALIDATION CRITERIA
- Mortality of parent test animals in the controls should not exceed 20 % at the end of the test.
- The mean number of living offspring in the control should be at least 60 per surviving adult Daphnia at the end of the test.
- The coefficient of variation around the mean number of living offspring produced per parent animal in the control should be ≤ 25 %.
- No ephippia are produced in the controls.
- Dissolved oxygen concentration should remain > 3 mg O2/L throughout the test.
- The pH of controls should be within the range 6-9 pH units and not vary by > 1.5 units throughout the test.
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
0.81 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: carbon content of test item
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
1.4 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: carbon content of test item
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
0.63 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: carbon content of test item
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.85 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: carbon content of test item
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.34 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: carbon content of test item
Basis for effect:
other: immobilisation and reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.69 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: carbon content of test item
Basis for effect:
other: immbolisation and reproduction
Duration:
21 d
Dose descriptor:
other: MATC
Effect conc.:
0.48 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: carbon content of test item
Basis for effect:
other: immobilisation and reproduction
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
3.7 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: boron content of test item
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
5.6 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: boron content of test item
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
3 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: boron content of test item
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
3.8 g/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: boron content of test item
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
1.9 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: boron content of test item
Basis for effect:
other: immobilisation and reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
3.3 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: boron content of test item
Basis for effect:
other: immobilisation and reproduction
Duration:
21 d
Dose descriptor:
other: MATC
Effect conc.:
2.5 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: boron content of test item
Basis for effect:
other: immobilisation and reproduction
Details on results:
CHEMICAL ANALYSIS OF TEST LOADING RATES
- Chemical analysis of the fresh test preparations on Days 0, 5, 9, 14 and 19 (see Appendix 4, attached) plus old test preparations on Days 2, 7, 12, 16 and 21 showed measured boron concentrations to range from less than the Limit of Quantification of the analytical method (determined to be 0.025 to 0.13 mg/L; equivalent to < LOQ to 6.15 mg/L based on test item boron content of 2.146 %).
- Equivalent test item concentrations based on the mean measured concentrations are shown in the table below.
- In instances where values of < LOQ were obtained, values of 0.5 LOQ were used for calculation purposes.

TOTAL ORGANIC CARBON ANALYSIS
- Total Organic Carbon (TOC) analysis of the fresh test preparations on Days 0, 5, 9, 14 and 19 (see Appendix 3, attached) plus old test preparations on Days 2, 7, 12, 16 and 21 showed measured carbon concentrations to range from less than the Limit of Quantification of the analytical method (determined to be 1.0 mg C/L; equivalent to < LOQ to 4.9 mg/L based on test item carbon content of 75.78 %).
- Given that values of < LOQ were obtained for many of the sampling points at the lower nominal concentrations, it was considered appropriate to calculate the equivalent time weighted mean measured test item concentration of the 5.0 % v/v saturated solution and calculate the remaining equivalent test item concentrations based on that value (see table below).
- In instances where values of < LOQ were obtained, values of 0.5 LOQ were used for calculation purposes.

VALIDATION CRITERIA
- Validation criteria achieved during the test are shown in the table below.

LETHAL EFFECTS ON THE PARENTAL GENERATION (P1)
- Mortality (immobilisation) occurred predominantly at the highest test concentration of 5.0 % v/v saturated solution and resulted in 50 % mortality by Day 14.
- A single mortality was also observed in the control group and the 1.25 % v/v saturated solution test group. However, these were considered to be due to natural causes and therefore not considered to have had an effect on the outcome of the test.
- ECx (immobilisation) values based on the equivalent test concentrations from TOC analysis were calculated by the Linear Interpolation method at 21 days and reported as EC10 (21 d) 0.81 mg/L and EC50 (21 d) 1.4 mg/L.
- ECx (immobilisation) values based on the equivalent test concentrations from the boron analysis were calculated by the Linear Interpolation method at 21 days and reported as EC10 (21 d) 3.7 mg/L and EC50 (21 d) 5.6 mg/L.
- It was not possible to determine 95 % confidence limits for the ECx immobilisation values due to the nature of the data.

SUB-LETHAL EFFECTS ON THE PARENTAL GENERATION (P1)
- There was a significant effect on size and colour or the daphnids in that surviving daphnids in the 5.0 % v/v saturated solution were markedly smaller and paler in colour than the control animals.
- On a few occasions throughout the test, parental daphnids in the 2.5 % v/v saturated solution test group were observed to be pale when compared to the control. However, these effects did not last past Day 16 and were therefore not considered to be due to prolonged exposure to the test item.
- Daphnids at the remaining test concentrations were observed to be the same size and colour as the control daphnids.
- After 21 days, the length of each surviving adult was determined and the results are shown in Table 9 (attached). There were no statistically significant differences (P ≥ 0.05) between the control and the 0.31, 0.63. 1.25 and 2.5 % v/v saturated solution test groups in terms of length of the daphnids after 21 days exposure to the test item (see Appendix 5, attached). However, a significant difference was observed in the length of the surviving Daphnia in the 5.0 % v/v saturated solution test group when compared to the control group.

EFFECTS ON REPRODUCTION
- After 21 days there were no statistically significant differences between the control and the 0.31, 0.63 and 1.25 % v/v satuarated solution test groups in terms of the number of live young produced per adult.
- The 2.5 and 5.0 % v/v saturated solution test groups showed a statistically significant difference from the control after 21 days in terms of producing fewer numbers of live young per adult (see Appendix 5, attached).
- ECx (reproduction) values based on equivalent test concentrations from the TOC analysis were calculated by the Maximum-Likelihood Probit method at 21 days and reported as EC10 (21 d) 0.63 mg/L and EC50 (21 d) 0.85 mg/L.
- ECx (reproduction values) based on equivalent test concentrations from the boron analysis were calculated by the Maximum-Likelihood Probit method at 21 days and reported as EC10 (21 d) 3.0 mg/L and EC50 (21 d) 3.8 mg/L.
- It was not possible to determine 95 % confidence limits for the ECx reproduction values due to the nature of the data.

EFFECTS ON THE FILIAL GENERATION (F1)
- Information on the effects of the test item on the F1 generation was limited since, by study design, the young were removed soon after liberation from the brood pouch. However, an assessment made at each media renewal showed the filial daphnids peoduced by the 0.31, 0.63, 1.25 and 2.5 % v/v saturated solution test groups were in the same general condition as the young produced by controls over the duration of the test.
- Young produced in the 5.0 % v/v saturated solution test concentration were observed on a number of occasions to be smaller and slower moving when compared to young produced by the control group.
- Young were first produced in the control test group on Day 9 of the investigation.
- There were no unhatched eggs or dead young in all control and treatment groups surviving to maturation.

LOWEST OBSERVED EFFECT CONCENTRATION
- The Lowest Observed Effect (LOEC) based on carbon content was 0.69 mg/L as this test group produced significantly fewer live young per adult (P < 0.05) than the control group.
- The Lowest Observed Effect (LOEC) based on boron content was 3.3 mg/L as this test group produced significantly fewer live young per adult (P < 0.05) than the control group.

NO OBSERVED EFFECT CONCENTRATION
- The No observed Effect Concentration (NOEC) based on carbon content was 0.34 mg/L as there were no significant mortalities (immobilisation) observed in the parental generation (P1) and there were no significant differences (P ≥ 0.05) in terms of the number of live young produced per adult when compared to the control after 21 days.
- The No observed Effect Concentration (NOEC) based on boron content was 1.9 mg/L as there were no significant mortalities (immobilisation) observed in the parental generation (P1) and there were no significant differences (P ≥ 0.05) in terms of the number of live young produced per adult when compared to the control after 21 days.

MAXIMUM ACCEPTABLE TOXICANT CONCENTRATION
- The Maximum Acceptable Toxicant Concentration (MATC) based on carbon content was calculated to be 0.48 mg/L.
- The Maximum Acceptable Toxicant Concentration (MATC) based on boron content was calculated to be 2.5 mg/L.

WATER QUALITY CRITERIA
- The results of water quality measurements are given in Table 10 (attached).
- Temperature was maintained at approximately 21 °C throughout the test. The results of hourly temperature measurement using a Testo temperature logger are shown in Figure 2 (attached).
- There were no treatment-related differences for oxygen concentration or pH.
- Water hardness was observed to be in the range 238 to 282 mg/L as CaCO3 in the control and the highest surviving test group throughout the test. Results of water hardness measurements are given in Table 11 (attached).
- Throughout the test, light intensity was observed to be in the range 510 to 677 lux (see Table 12, attached).

VORTEX DEPTH MEASUREMENTS
- The vortex depth was recorded at the start and end of the mixing period and was observed to have formed a dimple at the media surface.

OBSERVATIONS ON TEST ITEM SOLUBILITY
- Observations on the test media were carried out during mixing and testing of the saturated solution.
- At the start of the mixing period the 100 mg/L loading rate was observed to be a clear, colourless, water column with test item floating at the surface.
- After 23 hours stirring and a one hour standing period, the 100 mg/L loading rate was observed to remain as at the start of stirring.
- Microscopic inspection of the aqueous phase showed no micro-dispersions or undissolved test item to be present.
- After siphoning and for the duration of the test, the 0.31, 0.63, 1.25, 2.5 and 5.0 % v/v saturated solutions were observed to be clear, colourless, solutions.
Reported statistics and error estimates:
- The 21-day ECx (immobilisation) values and associated confidence limits were calculated by the Linear Interpolation method using the ToxCalc computer software package (ToxCalc; 1999).
- The ECx (reproduction) values and associated confidence limits after 21 days were calculated by the Maximum-Likelihood Probit method (Finney, 1977) using the ToxCalc computer software package (ToxCalc; 1999).
- For estimation of the Lowest Observed Effect Concentration (LOEC) and the No Observed Effect Concentration (NOEC), the numbers of live young produced per adult over the duration of the test were compared for the control and each test group using one way analysis of variance (Sokal and Rohlf, 1981) and the Williams test for differences between treatment means when several dose levels are compared with a zero dose control (Williams, 1971) (see Appendix 5, attached).
- Daphnia length data from the control and each test group, determined for the surviving daphnids on termination of the test, were 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) (see Appendix 5, attached).
- All statistical analyses were performed using the SAS computer software package (SAS 1999-2001).
- The Maximum Acceptable Toxicant Concentration (MATC) was calculated using the geometric mean method where MATC = square route of NOEC x LOEC and NOEC = No Observed Effect Concentration (mg/L) ; LOEC = Lowest Observed Effect Concentration (mg/L).

EQUIVALENT TEST ITEM CONCENTRATIONS BASED ON MEAN MEASURED BORON CONCENTRATIONS

Nominal concentration (% v/v saturated solution)

Equivalent test item concentration (mg/L)

0.31

0.74

0.63

0.92

1.25

1.87

2.5

3.25

5.0

5.64

EQUIVALENT TEST ITEM CONCENTRATIONS BASED ON TIME WEIGHTED MEAN MEASURED CONCENTRATION OF CARBON IN THE 5.0 % v/v SATURATED SOLUTION TEST GROUP

Nominal concentration (% v/v saturated solution)

Equivalent test item concentration (mg/L

0.31

0.085

0.63

0.17

1.25

0.34

2.5

0.69

5.0

1.37

VALIDATION CRITERIA

 

Required

Actual

Control mortality

≤ 20 %

10 %

Mean number of live young per surviving adult (control group)

≥ 60 after 21 days

108

Coefficient of variation for control group

≤ 25 %

21 %

Ephippia produced

0

0

Dissolved oxygen

> 3 mg O2/L

≥ 6.2 mg O2/L

pH (control group)

Deviation ≤ 1.5

Deviation 1.3

pH range (control group)

6-9

7.3 to 8.6

Validity criteria fulfilled:
yes
Conclusions:
Exposure of Daphnia magna to the test item resulted in significant mortalities at the nominal test concentration of 5.0 % v/v saturated solution resulting in 50 % mortalities by Day 14. The 21-day EC50 (immobilisation) value was estimated to be 1.4 mg/L based on time weighted mean measured test concentrations of carbon content for the parental Daphnia generation (P1). The 21-day EC50 (immobilisation) value was estimated to be 5.6 mg/L based on mean measured test concentrations of boron content for the parental Daphnia generation (P1). The 21-day EC50 (reproduction) value was estimated to be 0.85 mg/L based on time weighted mean measured test concentrations of carbon content (95 % confidence limits could not be calculated due to the nature of the data). The 21-day EC50 (reproduction) value was estimated to be 3.8 mg/L based on mean measured test concentrations of boron content (95 % confidence limits could not be calculated due to the nature of the data). For the test concentration based on carbon content, the Lowest Observed Effect Concentration (LOEC) was 0.69 mg/L and the No Observed Effect Concentration (NOEC) was 0.34 mg/L. The maximum Acceptable Toxicant Concentration (MATC) was calculated to be 0.48 mg/L. For the test concentration based on boron content, the Lowest Observed Effect Concentration (LOEC) was 3.3 mg/L and the No Observed Effect Concentration (NOEC) was 1.9 mg/L. The maximum Acceptable Toxicant Concentration (MATC) was calculated to be 2.5 mg/L.
Executive summary:

GUIDELINE

A study was performed to assess the chronic toxicity of the test item to Daphnia magna. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2012) No 211 “Daphnia magna Reproduction Test” referenced as Method C.20 of Commission Regulation (EC) No 440/2008 and the US EPA Draft Ecological Effects Test Guideline OPPTS 850.1300 “Daphnid Chronic Toxicity Test”.

 

METHODS

Due to the low aqueous solubility and pure nature of the test item, the medium was prepared as a slow stir saturated solution for the definitive test. Based on the results of an acute toxicity study, Daphnia magna were exposed (10 replicates of a single daphnid per group) to a solution of the test item over a range of test concentrations (0.31, 0.63, 1.25, 2.5 and 5.0 % v/v saturated solution) for a period of 21 days. The test solutions were renewed three times per week during the test. The number of live and dead adult Daphnia and young daphnids (live and dead) were determined daily. The Daphnia were fed daily with a mixture of algal suspension and Tetramin flake food suspension.

 

RESULTS

Chemical analysis of the fresh test preparations on days 0, 5, 9, 14 and 19 plus the old test preparations on days 2, 7, 12, 16 and 21 showed boron concentrations to range from less than the Limit of Quantification (LQQ) for the analytical method (determined to be 0.025 mg/L) to 0.13 mg/L (equivalent to test item concentrations of less than the LQQ to 6.15 mg/L based on test item boron content of 2.146 %). Total Organic Carbon (TOC) analysis of the fresh test preparations on days 0, 5, 9, 14 and 19 plus the old test preparations on days 2, 7, 12, 16 and 21 showed measured carbon concentrations to range from less than the LQQ of the analytical method (assessed as being 1.0 mg C/L) to 3.7 mg/L (equivalent to test item concentrations of less than the LQQ to 4.9 mg/L based on test item carbon content of 75.78 %). Given that values of less than the LQQ were obtained for many of the sampling points at the lower nominal concentrations, it was considered appropriate to calculate the equivalent time weighted mean measured test item concentration of the 5.0 % v/v saturated solution and calculate the remaining equivalent test concentrations based on that value.

 

CONCLUSION

Exposure of Daphnia magna to the test item resulted in significant mortalities at the nominal test concentration of 5.0 % v/v saturated solution resulting in 50 % mortalities by Day 14. Based on equivalent test item concentrations from Total Organic Carbon (TOC) analysis, exposure of Daphnia magna to the test item gave EC10 (21 d) 0.81 mg/L and EC50 (21 d) 1.4 mg/L based on immobilisation. The EC10 (21 d) was 0.63 mg/L and the EC50 (21 d) value was 0.85 mg/L based on reproduction. The No Observed Effect Concentration (NOEC) was reported as 0.34 mg/L, the Lowest Observed Effect Concentration (LOEC) was 0.69 mg/L and the Maximum Acceptable Toxicant Concentration (MATC) was 0.48 mg/L. Based on equivalent test item concentrations from boron analysis, exposure of Daphnia magna to the test item gave EC10 (21 d) 3.7 mg/L and EC50 (21 d) 5.6 mg/L based on immobilisation. The EC10 (21 d) was 3.0 mg/L and the EC50 (21 d) value was 3.8 mg/L based on reproduction. The No Observed Effect Concentration (NOEC) was reported as 1.9 mg/L, the Lowest Observed Effect Concentration (LOEC) was 3.3 mg/L and the Maximum Acceptable Toxicant Concentration (MATC) was 2.5 mg/L.

Description of key information

Exposure of Daphnia magna to the test item resulted in significant mortalities at the nominal test concentration of 5.0 % v/v saturated solution resulting in 50 % mortalities by Day 14. Based on equivalent test item concentrations from Total Organic Carbon (TOC) analysis, exposure of Daphnia magna to the test item gave EC10 (21 d) 0.81 mg/L and EC50 (21 d) 1.4 mg/L based on immobilisation. The EC10 (21 d) was 0.63 mg/L and the EC50 (21 d) value was 0.85 mg/L based on reproduction. The No Observed Effect Concentration (NOEC) was reported as 0.34 mg/L, the Lowest Observed Effect Concentration (LOEC) was 0.69 mg/L and the Maximum Acceptable Toxicant Concentration (MATC) was 0.48 mg/L. Based on equivalent test item concentrations from boron analysis, exposure of Daphnia magna to the test item gave EC10 (21 d) 3.7 mg/L and EC50 (21 d) 5.6 mg/L based on immobilisation. The EC10 (21 d) was 3.0 mg/L and the EC50 (21 d) value was 3.8 mg/L based on reproduction. The No Observed Effect Concentration (NOEC) was reported as 1.9 mg/L, the Lowest Observed Effect Concentration (LOEC) was 3.3 mg/L and the Maximum Acceptable Toxicant Concentration (MATC) was 2.5 mg/L.(OECD 211, EU Method C.20 and OPPTS 850.1300).

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
1.9 mg/L

Additional information

GUIDELINE

A study was performed to assess the chronic toxicity of the test item to Daphnia magna. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2012) No 211 “Daphnia magna Reproduction Test” referenced as Method C.20 of Commission Regulation (EC) No 440/2008 and the US EPA Draft Ecological Effects Test Guideline OPPTS 850.1300 “Daphnid Chronic Toxicity Test”.

 

METHODS

Due to the low aqueous solubility and pure nature of the test item, the medium was prepared as a slow stir saturated solution for the definitive test. Based on the results of an acute toxicity study, Daphnia magna were exposed (10 replicates of a single daphnid per group) to a solution of the test item over a range of test concentrations (0.31, 0.63, 1.25, 2.5 and 5.0 % v/v saturated solution) for a period of 21 days. The test solutions were renewed three times per week during the test. The number of live and dead adult Daphnia and young daphnids (live and dead) were determined daily. The Daphnia were fed daily with a mixture of algal suspension and Tetramin flake food suspension.

 

RESULTS

Chemical analysis of the fresh test preparations on days 0, 5, 9, 14 and 19 plus the old test preparations on days 2, 7, 12, 16 and 21 showed boron concentrations to range from less than the Limit of Quantification (LQQ) for the analytical method (determined to be 0.025 mg/L) to 0.13 mg/L (equivalent to test item concentrations of less than the LQQ to 6.15 mg/L based on test item boron content of 2.146 %). Total Organic Carbon (TOC) analysis of the fresh test preparations on days 0, 5, 9, 14 and 19 plus the old test preparations on days 2, 7, 12, 16 and 21 showed measured carbon concentrations to range from less than the LQQ of the analytical method (assessed as being 1.0 mg C/L) to 3.7 mg/L (equivalent to test item concentrations of less than the LQQ to 4.9 mg/L based on test item carbon content of 75.78 %). Given that values of less than the LQQ were obtained for many of the sampling points at the lower nominal concentrations, it was considered appropriate to calculate the equivalent time weighted mean measured test item concentration of the 5.0 % v/v saturated solution and calculate the remaining equivalent test concentrations based on that value.

 

CONCLUSION

Based on equivalent test item concentrations from Total Organic Carbon (TOC) analysis, exposure of Daphnia magna to the test item gave EC10 (21 d) 0.81 mg/L and EC50 (21 d) 1.4 mg/L based on immobilisation. The EC10 (21 d) was 0.63 mg/L and the EC50 (21 d) value was 0.85 mg/L based on reproduction. The No Observed Effect Concentration (NOEC) was reported as 0.34 mg/L, the Lowest Observed Effect Concentration (LOEC) was 0.69 mg/L and the Maximum Acceptable Toxicant Concentration (MATC) was 0.48 mg/L. Based on equivalent test item concentrations from boron analysis, exposure of Daphnia magna to the test item gave EC10 (21 d) 3.7 mg/L and EC50 (21 d) 5.6 mg/L based on immobilisation. The EC10 (21 d) was 3.0 mg/L and the EC50 (21 d) value was 3.8 mg/L based on reproduction. The No Observed Effect Concentration (NOEC) was reported as 1.9 mg/L, the Lowest Observed Effect Concentration (LOEC) was 3.3 mg/L and the Maximum Acceptable Toxicant Concentration (MATC) was 2.5 mg/L.