Oxirane, mono[(C12-14-alkyloxy)methyl] derivs.

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 29 September 2016 and 06 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2012

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Version / remarks:

EC NO. 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Specific details on test material used for the study:

Identification : Oxirane, mono [(C12-14-alkyloxy) methyl] derivs
Physical state/Appearance: Clear colourless liquid
Batch : AAD1326100
Purity : 100% UVCB
Expiry Date : 29 August 2019
Storage Conditions: Stored at room temperature in the dark between 25 June 2016 and 29 June 2016 and then stored cold at ~ 4 °C in the dark thereafter

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples of the fresh test preparations were taken on Days 0, 5, 9, 14 and 19 and of the expired test preparations on Days 2, 7, 13, 16 and 21. Samples were stored frozen prior to analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

Range-finding Test:
Nominal amounts of test item (23 and 228 μl, equivalent to 20 and 200 mg) were each separately added to the surface of 2 liters of test water to give the 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. 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 75-100 mL discarded) to give the 10 and 100 mg/L loading rate WAFs.
A sample of each test concentration was taken for chemical analysis on Days 0 and 3 in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed

Definitive Test:
Nominal amounts of test item (57, 102, 182, 319 and 570 μl, equivalent to 50, 90, 160, 280 and 500 mg) were each separately added to the surface of 5 liters of test water to give the 10, 18, 32, 56 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 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, however, glass wool was used as a precautionary measure. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 10, 18, 32, 56 and 100 mg/L loading rate WAFs.
The concentration and stability of the test item in the test preparations were verified by chemical analysis on Days 0, 2, 5, 7, 9, 13, 14, 16, 19 and 21

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

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 Appendix 1) in a temperature controlled room at approximately 20”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.

Study design

Test type:

static

Water media type:

other: The reconstituted water (Elendt M7 medium) used for the range-finding and definitive tests was the same as that used to maintain the stock animals

Limit test:

no

Total exposure duration:

21 d

Test conditions

Hardness:

246 to 262 mg/L as CaCO3

Test temperature:

19 - 22”C

pH:

7.9 ± 0.3

Dissolved oxygen:

8.3 - 9.1 mg O2/L

Nominal and measured concentrations:

the results were based on nominal loading rates only.

Details on test conditions:

Experimental Design and Study Conduct
Range-finding Test
The test concentrations to be used in the definitive test 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 10 and 100 mg/L.
Nominal amounts of test item (23 and 228 μl, equivalent to 20 and 200 mg) were each separately added to the surface of 2 liters of test water to give the 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. 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 75-100 mL discarded) to give the 10 and 100 mg/L loading rate WAFs.
In the range-finding test, 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 five replicate test vessels were prepared. The water temperature was maintained 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 10 days. The control group was maintained under identical conditions but not exposed to the test item.
The test preparations were renewed on Days 3, 5 and 7. The adult daphnia were transferred to fresh media by wide-bore pipette before the contents of each vessel were 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 20 µL of Tetramin® flake food suspension daily. Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid with the exception of Control replicate 2 on Day 4 which received extra tetramin suspension in error.
A sample of each test concentration was taken for chemical analysis on Days 0 and 3 in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.

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 Water Accommodated Fraction (WAF) of the test item over a range of test concentrations of 10, 18, 32, 56 and 100 mg/L loading rate WAF for a period of 21 days. The test solutions were renewed 3 times per week.
Nominal amounts of test item (57, 102, 182, 319 and 570 μl, equivalent to 50, 90, 160, 280 and 500 mg) were each separately added to the surface of 5 liters of test water to give the 10, 18, 32, 56 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 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, however, glass wool was used as a precautionary measure. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 10, 18, 32, 56 and 100 mg/L loading rate WAFs.
The concentration and stability of the test item in the test preparations were verified by chemical analysis on Days 0, 2, 5, 7, 9, 13, 14, 16, 19 and 21.

Exposure Conditions
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 18 to 22”C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light (not exceeding 1500 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods for 21 days. The test vessels were not aerated. The diluent water only was aerated prior to use.
The control group was maintained under identical conditions but not exposed to the test item.
The test preparations were renewed 3 times per week on Days 2, 5, 7, 9, 13, 14, 16, and 19. The adult daphnia were transferred to fresh media by wide-bore pipette before the contents of each vessel were 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 20 µL of Tetramin® flake food suspension daily. Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.

Assessments

Test Organism Observations
On a daily basis the numbers of live and dead of 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. Adult daphnia which were unable to swim for approximately 15 seconds after gentle agitation (ie. immobile), were considered to be dead. An immobilization criterion for the young daphnids was considered to be inappropriate due to the large numbers of off-spring 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 whilst the 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.
The water hardness of the control and the highest surviving test concentration in the fresh and old media was measured once per week using the methods described in Fields and On-Site Methods for Analysis of Water (British Standards Institution, 1993).

Verification of Test Concentrations
Water samples were taken from the control and each surviving test group (replicates pooled) for quantitative analysis. Samples of the fresh test preparations were taken on Days 0, 5, 9, 14 and 19 and of the expired test preparations on Days 2, 7, 13, 16 and 21. Samples were stored frozen prior to analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.

Data Evaluations
ELx Calculations
The 21-Day ELx (immobilization) values and associated confidence limits were calculated by binomial method with associated confidence limits set as the concentrations resulting in 0% and 100% immobilization
The ELx (reproduction) values and associated confidence limits after 21 days were calculated by the 3-parameter normal cumulative distribution function method using the ToxRat Professional computer software package (TOXRAT).

NOEL and LOEL Determinations
For the estimation of the "Lowest Observed Effect Loading Rate" (LOEL) and the "No Observed Effect Loading Rate " (NOEL), the numbers of live young produced per adult over the duration of the test for the each test group were compared using the Multiple Sequentially-rejective Median (2x2-Table) Test after Bonferroni-Holm.
Results from the control and 10, 18, 32 and 56 mg/L loading rate WAF test groups daphnia length data, determined for the surviving daphnids on termination of the test, were compared using Multiple Sequentially-rejective Median (2x2-Table) Test after Bonferroni-Holm The 100 mg/L loading rate WAF test group data were not included in the statistical analysis as the effects of exposure eliminated all the daphnids prior to termination of the test.
All NOEL and LOEL determinations were conducted using the ToxRat Professional computer software package (TOXRAT).

Validation Criteria
For the test to be valid, the following criteria should be fulfilled:
• 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 equal to or less than 25%.
• No ephippia are produced in the controls.
• Dissolved oxygen concentration should remain greater than 3 mg O2/L throughout the test.
• pH of the controls should be within the range 6 to 9 pH units and not vary by greater than 1.5 units throughout the test.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

75 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Effect concentrations 2

Key result

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

64 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 3

Key result

Duration:

21 d

Dose descriptor:

LOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Effect concentrations 4

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Details on results:

Range-finding Test
Cumulative immobilization and sub-lethal effects data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1.
No immobilization was observed in any of the test concentrations. Sub-lethal effects of exposure were observed at the test concentrations of 100 mg/L loading rate WAF. The response observed was a reduction in the number of young produced.
Chemical analysis of the test preparations at 0 and 48 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.047 mg/L. This does not infer that no test item was in solution, just that any dissolved test item present was at a concentration of less than the LOQ.
Based on this information test concentrations of 10, 18, 32, 56 and 100 mg/L loading rate WAF were selected for the definitive test.

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 Water Accommodated Fraction (WAF) of the test item over a range of test concentrations of 10, 18, 32, 56 and 100 mg/L loading rate WAF for a period of 21 days. The test solutions were renewed 3 times per week throughout the test.

Verification of Test Concentrations
Chemical analysis of the test preparations showed that in the majority of samples, measured concentrations were less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.047 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was present as a concentration below the LOQ.
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.

Test Organism Observations
The observations for each test and control group are summarized in Table 2. The number of live young produced per adult is shown in Table 3 and the total cumulative production of live young is given in Table 4. .

One daphnia in the 18 mg/L loading rate WAF group was observed as being pale in color and small, produced no live young and was considerably smaller than all other parental daphnia. Inclusion of data from this replicate did not induce a statistically significant effect in terms of reproduction or body length and all results reported include data from this replicate.

Validation Criteria
The following validation criteria were achieved during the test

Required Actual
a) Control mortality < 20% 0%
b) Mean number of live youngper
surviving adult (control group) ≥ 60 after 21 days 135
c) Coefficient of variation for
control group < 25% 8.4%
d) Ephippia produced 0 0
e) Dissolved oxygen > 3 mg O2/L ≥7.9 mg O2/L
f) pH (control group) deviation >1.5 0.3

Lethal Effects on the Parental Generation (P1)
Mortality (immobilization) occurred predominantly at the highest test loading rate of 100 mg/L resulting in 100% mortality by Day 4. Mortality was also observed at the test loading rates of 10 mg/L on Day 16 and Day18, in 18 mg/L on Day 19 and in 32 mg/L on Day 6, however, statistical analysis of the mortality data using the Fisher’s Exact test showed that the observed mortalities in the 10, 18 and 32 mg/L loading rate WAF groups were not significantly different (P0.05) when compared to the control group and therefore these mortalities are considered to be inadvertent mortalities. No mortalities occurred at the 56 mg/L loading rate WAF group throughout the test.
The following ELx (immobilization) values based on nominal loading rates were calculated by the Linear Interpolation method at 21 days:

Endpoint Concentration (mg/L Loading Rate WAF)
Immobilization EL10 n.d.
EL50 75
95% confidence limits 56 - 100

Sub-lethal Effects on the Parental Generation (P1)
After 21 days the length of each surviving adult was determined, the results of which are given in Table 5. The results showed that there were no statistically significant differences (P≥ 0.05) between the control and the 10, 18, 32 and 56 mg/L test groups in terms of length of the daphnids after 21 days exposure to the test item. The 100 mg/L loading rate WAF group data was not included in this analysis as exposure to the test item eliminated all the daphnids prior to Day 21 of the test.

Effects on Reproduction
After 21 days, there were no statistically significant differences between the control and the 10, 18, 32 and 56 mg/L loading rate WAF groups in terms of the number of live young produced per adult, however, the 100 mg/L test group showed a statistically significant difference from the control after 21 days in terms of producing fewer numbers of live young per adult.
The following ELx (reproduction) values based on nominal test concentrations were estimated from inspection of the immobilization data at 21 days:

Endpoint Concentration (mg/L Loading Rate WAF)
Reproduction EL10 58
95% confidence limits n.d.
EL50 64
95% confidence limits n.d.

Effects on the Filial Generation (F1)
Information on the effects of the test item on the F1 generation is limited, since, by study design, the young are removed soon after liberation from the brood pouch. However, an assessment made at each media renewal showed the "filial" daphnids produced by all the test groups were in the same general condition as the young produced by the controls over the duration of the test.
Young were first produced in the control test group on Day 8 of the test.
Due to the toxic effect of the test item the parental generation (P1) of the 100 mg/L loading rate WAF group were eliminated prior to the production of young.
There were no unhatched eggs or dead young observed in any of the control and treatment groups.

Lowest Observed Effect Loading Rate
The "Lowest Observed Effect Loading Rate" (LOEL) was 100 mg/L as this test group did not produce any live young due to the toxic effect of the test item eliminating the parental generation by Day 4 of the test.

No Observed Effect Loading Rate
The "No Observed Effect Loading Rate" (NOEL) was 56 mg/L as there were no significant mortalities (immobilization) 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.

Water Quality Criteria
The results of the water quality measurements are given in Table 6. Temperature was maintained at approximately 19”C to 22”C throughout the test, while there were no treatment related differences for oxygen concentration or pH. The water temperature was also recorded in the control vessel every hour using a Testo temperature logger.
The water hardness was observed to be in the range 246 to 262 mg/L as CaCO3 in the control and the highest surviving test group throughout the test.
Throughout the test the light intensity was observed to be in the range 309 to 532 lux (see Table 8).
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 the mixing and testing of the WAFs.
At the start of the mixing periods the 10, 18, 32, 56 and 100 mg/L loading rates were observed to be a clear colourless water column with globules of test item floating on the surface of the water.
After 23 hours stirring and a 1-Hour standing period all loading rates were observed to be clear colourless water columns with test item floating on the surface of the water. Microscopic inspection of the 10, 18, 32 and 56 mg/L loading rates showed no micro-dispersions or undissolved test item to be present, however, on occasions the 100 mg/L loading rate showed micro-dispersions or undissolved test item to be present. As a precautionary measure, all WAFs were siphoned through a glass wool plug prior to dispensing. For the duration of the test, the 10, 18, 32, 56 and 100 mg/L loading rates were observed to be clear, colourless solutions.

Any other information on results incl. tables

Table1            Range-finding TestData

Nominal Loading Rate (mg/L)	Percentage Immobilization After 10 Days Exposure	Sub-lethal Effects	Total Number of Live Young in First Brood
Control	0	None	55
10	0	None	55
100	0	None	42

Table 2 Summary of Findings Following the Exposure ofDaphnia magnafor 21 Days

Nominal Loading Rate (mg/L)	Percentage Survival of Parental (P1) Generation	Total Number of Live Young	Number of Live Young Per Parent Surviving to the End of the Test	Number of Live Young Per Parent at the Start of the Test Excluding Parental Accidental and/or Inadvertent Mortalities
Control	100	1351	135	135
10	80	1240	130	130
18	90	1098	112	112
32	80	1237	140	140
56	100	1248	125	125
100	0	0	0	0

Table3            Summary of Daily Observations

Day	Nominal Loading Rate (mg/L)
	Control		10		18		32		56		100
	Adults Surviving	Live Young	Adults Surviving	Live Young	Adults Surviving	Live Young	Adults Surviving	Live Young	Adults Surviving	Live Young	Adults Surviving	Live Young
1	10	0	10	0	10	0	10	0	10	0	9	0
2	10	0	10	0	10	0	10	0	10	0	5	0
3	10	0	10	0	10	0	10	0	10	0	1	0
4	10	0	10	0	10	0	10	0	10	0	0	0
5	10	0	10	0	10	0	10	0	10	0	0	0
6	10	0	10	0	10	0	9	0	10	0	0	0
7	10	0	10	0	10	0	9	0	10	0	0	0
8	10	92	10	114	10	80	9	124	10	113	0	0
9	10	0	10	0	10	0	9	1	10	0	0	0
10	10	0	10	0	10	16	9	0	10	0	0	0
11	10	201	10	194	10	147	9	223	10	193	0	0
12	10	1	10	5	10	10	9	1	10	3	0	0
13	10	0	10	29	10	10	9	171	10	85	0	0
14	10	279	10	266	10	223	9	77	10	192	0	0
15	10	0	10	0	10	25	9	0	10	0	0	0
16	10	178	9	164	10	246	9	326	10	307	0	0
17	10	213	9	184	10	32	9	0	10	42	0	0
18	10	0	9	0	10	30	9	0	10	0	0	0
19	10	349	8	240	9	279	8	314	10	311	0	0
20	10	36	8	44	9	0	8	0	10	2	0	0
21	10	2	8	0	9	0	8	0	10	0	0	0
TOTALS	10	1351	8	1240	9	1098	8	1237	10	1248	0	0

Table4            Total Cumulative Production of Live Young

Nominal Loading Rate (mg/L)	Day
	1	2	3	4	5	6	7	8	9	10	11
Control	0	0	0	0	0	0	0	92	92	92	293
10	0	0	0	0	0	0	0	114	114	114	308
18	0	0	0	0	0	0	0	80	80	96	243
32	0	0	0	0	0	0	0	124	125	125	348
56	0	0	0	0	0	0	0	113	113	113	306
100	0	0	0	0	0	0	0	0	0	0	0

 

Nominal Loading Rate (mg/L)	Day
	12	13	14	15	16	17	18	19	20	21
Control	294	294	573	573	751	964	964	1313	1349	1351
10	313	342	608	608	772	956	956	1196	1240	1240
18	253	263	486	511	757	789	819	1098	1098	1098
32	349	520	597	597	923	923	923	1237	1237	1237
56	309	394	586	586	893	935	935	1246	1248	1248
100	0	0	0	0	0	0	0	0	0	0

 

Table 5            Body Length of Surviving Adults at Day 21

Nominal Loading Rate (mg/L)	Individual Daphnia Lengths (mm)
	1	2	3	4	5	6	7	8	9	10
Control	4.2	4.4	4.1	4.3	4.2	4.3	4.1	4.4	4.2	4.1
10	-	4.3	-	4.3	4.3	4.2	4.2	4.1	4.1	4.1
18	4.1	4.3	4.2	4.2	4.1	-	4.2	4.2	4.1	2.3
32	-	-	4.3	4.2	4.1	4.2	4.2	4.3	4.3	4.2
56	4.1	4.3	4.2	4.2	4.2	4.2	4.3	4.0	4.1	4.0
100	-	-	-	-	-	-	-	-	-	-

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Daphnia magna to the test item resulted in significant mortalities at the loading rate of 100 mg/L resulting in 100 mortalities by Day 4.
The 21-Day EL50 (immobilization) value, based on nominal loading rates, for the parental daphnia generation (P1) was calculated to be 75 mg/L with 95% confidence limits of 56 to 100 mg/L.
The 21-Day EL50 (reproduction) based on nominal loading rates was 64 mg/L.
The "Lowest Observed Effect Loading Rate" (LOEL) and the "No Observed Effect Loading Rate" (NOEL) based on nominal loading rates were 100 and 56 mg/L respectively.

Executive summary:

A study was performed to assess the chronic toxicity of the test item toDaphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2012) No 211, "DaphniamagnaReproduction Test" referenced as Method C.20 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).

Based on the results of a preliminary range-finding test, Daphnia magnawere exposed
(10 replicates of a single daphnid per group) to a Water Accommodated Fraction (WAF) of the test item over a range of test concentrations of 10, 18, 32, 56 and 100 mg/L for a period of 21 days. The test solutions were renewed 3 times per week

The numbers 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 test preparations showed that in the majority of samples, measured concentrations were less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.047 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was present as a concentration below the LOQ.

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.

Exposure ofDaphnia magnato the test item gave the following results based on the nominal loading rates:

Endpoint		Concentration (mg/L Loading Rate WAF)
Immobilization	EL10	n.d.
	EL50	75
	95% confidence limits	56 - 100
Reproduction	EL10	58
	95% confidence limits	n.d.
	EL50	64
	95% confidence limits	n.d.
No Observed Effect Loading Rate (NOEL)		56
Lowest Observed Effect Loading Rate (LOEL)		100

nd. = not determined for mathematical reasons