Reaction products of 1-(substitutedphenyl)urea coupled with diazotated potassium sodium substituted-5-{[2-(substituted)ethyl]sulfonyl}benzenesulfonate, further condensed with 2,4,6-trichloro-1,3,5-triazine, further converted with disubstituted benzene-1,4-disulfonic acid in aq. sodium hydroxide

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 12 October and 24 November 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

referenced as Method C.20 of Commission Regulation (EC) No. 440/2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Water samples were taken from the control and each test group for quantitative analysis. Samples of the fresh test preparations were taken from the bulk test preparations on Days 0, 5, 9, 14 and 19, and of the expired test preparations (replicates pooled) 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.

Vehicle:

yes

Remarks:

test water

Details on test solutions:

RANGE-FINDING TEST:
Daphnia magna were exposed to a series of nominal concentrations of 0.10, 1.0, 10 and 100 mg/L.

A nominal amount of test item (100 mg) was dissolved in test water and the volume adjusted to 1 liter to give the 100 mg/L test concentration from which a series of dilutions was made to give the required test concentrations of 0.10, 1.0 and 10 mg/L. Each of the prepared concentrations was inverted several times to ensure adequate mixing and homogeneity



DEFINITIVE TEST:
A nominal amount of test item (400 mg) was dissolved in test water,reconstituted water (Elendt M7 medium), and the volume adjusted to 2 liters to give a 200 mg/L stock solution. A series of dilutions was made from this stock solution to give the required test concentrations of 10, 18, 32, 56 and 100 mg/L.
The stock solution and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis on Days 0, 2, 5, 7, 9, 12, 14, 16, 19 and 21.

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 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.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

None

Hardness:

234 to 247 mg/L CaCO3

Test temperature:

18 to 22 °C with a maximum deviation of ±1 °C

pH:

7.7 to 8.1

Dissolved oxygen:

≥7.7 mg O2/L

Salinity:

Not applicable

Conductivity:

No data

Nominal and measured concentrations:

RANGE-FINDING TEST:
Nominal concentrations: 0.1, 1.0, 10 and 100 mg/L


DEFINITIVE TEST:
Nominal concentrations: 10, 18, 32, 56 and 100 mg/L.
Analysis of the fresh and old test preparations on days 0, 2, 5, 7, 9, 12, 14, 16, 19 and 21 showed measured test concentrations were all near nominal therefore the test results are based on nominal test concentrations only.

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 mL glass vessels
- Type (delete if not applicable): closed
- Fill volume: 100mL
- Aeration: The diluent water was aerated prior to use, however, the test vessels were not aerated.
- Renewal rate of test solution (frequency/flow rate): The test preparations were renewed 3 times per week on Days 2, 5, 7, 9, 12, 14, 16 and 19
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M7 medium
- Culture medium different from test medium: No


OTHER TEST CONDITIONS
- Photoperiod: 16 hours light, 8 hours darkness with 20 minute dawn and dusk transition periods
- Light intensity: Throughout the test the light intensity was observed to be in the range 522 to 771 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
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.
Filial daphnids were considered to be dead if no sign of movement was apparent during examination. Parental 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.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L.
- Results used to determine the conditions for the definitive study: No immobilization was observed at the test concentrations of 0.10, 10 and 100 mg/L, however, immobilization was observed at 1.0 mg/L. Sub-lethal effects of exposure were observed at the test concentration of 100 mg/L. This response was a delayed and reduced size of first brood and aborted eggs were also observed. Based on this information test concentrations of 10, 18, 32, 56 and 100 mg/L were selected for the definitive test.

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

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

60 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

84 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

75 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% confidence limits: 29-85 mg/L

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

94 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% confidence limits: 76-96 mg/L

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

other: MATC

Effect conc.:

75 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Geometric mean between NOEC and LOEC

Details on results:

Range-finding Test
No immobilization was observed at the test concentrations of 0.10, 10 and 100 mg/L, however, immobilization was observed at 1.0 mg/L. Sub-lethal effects of exposure were observed at the test concentration of 100 mg/L. This response was a delayed and reduced size of first brood and aborted eggs were also observed.

Based on this information test concentrations of 10, 18, 32, 56 and 100 mg/L were selected for the definitive test.

Chemical analysis of the test preparations on Days 0 and 2 showed test concentrations to range from 101% to 112% of the nominal concentration, indicating that the test item was stable under test conditions.

Definitive Test
Based on the results of preliminary range finding test, test concentrations of 10, 18, 32, 56 and 100 mg/L were selected for the definitive test.

Verification of Test Concentrations
Analysis of the fresh test preparations on days 0, 5, 9, 14 and 19 showed measured test concentrations to range from 89% to 101% of nominal concentrations. There was no significant change in the measured concentrations of the aged test preparations on days 2, 7, 12, 16 and 21, so the results are based on nominal test concentrations only.

Validation Criteria
The following validation criteria were achieved during the test
Required Actual
Control mortality <=20% 0%
Mean number of live young per surviving adult (control group) >=60 after 21 days 116
Coefficient of variation for control group <=25% 7.7%
No ephippia produced (control group) 0 produced 0 produced
Dissolved oxygen > 3 mg O2/L >=7.7 mg O2/L
pH (control group) 6 to 9 7.7 to 8.1
Variation <=1.5 0.5

Lethal Effects on the Parental Generation (P1)
Mortality (immobilization) occurred predominantly at the highest test concentration of 100 mg/L resulting in 78% mortality by Day 19. Mortality also occurred in the 10 mg/L test concentration, resulting in 10% mortality by Day 21, however due to the lack of a concentration dose response this was considered to be an inadvertent mortality. No mortalities occurred at the 18, 32 and 56 mg/L test groups throughout the test.

Sub-lethal Effects on the Parental Generation (P1)
Five of the surviving daphnia in the 100 mg/L test concentration were observed to be pale in appearance compared to the control on Day 17 of the test. Three of the pale colored daphnia were mortalities on Day 19 of the test whereas the remaining two survived to the end of the test but remained pale in color.
The daphnids at the remaining test concentrations were observed to be the same size and color as the control daphnids.
After 21 days the length of each surviving adult was determined, the results of which are given in Table 10. The reduction in daphnia length at the test concentrations of 10, 18, 32, 56 and 100 mg/L were -0.3%, 1.2%, 3.4%, 4.6% and 13% respectively. The results showed that there were no statistically significant differences (P≥ 0.05) between the control and the 10 and 18 mg/L test groups in terms of length of the daphnids after 21 days exposure to the test item, however there was a significant difference between the control and the 32, 56 and 100 mg/L test groups.
Given that the percentage reduction in body length at 32 and 56 mg/L were less than 5% and therefore below the calculated EC10 it was considered that the observed reductions in body length although statistically significant were not necessarily biologically significant. The NOEC and LOEC for body length after 21 days are therefore considered to be 56 mg/L and 100 mg/L respectively by inspection of the data.

Effects on Reproduction
After 21 days there were no statistically significant differences between the control and the 10, 18, 32 and 56 mg/L test groups in terms of the number of live young produced per adult. 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.

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 the 10 and 32 mg/L test groups were in the same general condition as the young produced by the controls over the duration of the test. The young produced by replicate 3 of the 18 mg/L test group were observed to be pale when compared to the control young on Days 16. The young produced by replicate 6 of the 56 mg/L test group were observed to be small and pale when compared to the control group on day 19 and the young produced by the 100 mg/L test group were observed to be small and pale when compared to the control group between days 17 and 21.
Young were first produced in the control test group on Day 7 of the test.
There were no unhatched eggs or dead young recorded in any of the control and treatment groups.

Lowest Observed Effect Concentration
The "Lowest Observed Effect Concentration" (LOEC) was 100 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) 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.

Maximum Acceptable Toxicant Concentration
The "Maximum Acceptable Toxicant Concentration" (MATC) was calculated to be 75 mg/L.

Observations on Test Item Solubility
At the start and throughout the test all control solutions were observed to be clear colorless, the 10 mg/L solutions were bright yellow, the 18 mg/L solutions were dark yellow, the 32 mg/L solutions were light orange, the 56 mg/Lsolutions were bright orange and the 100 mg/L solutions were dark orange.

95% confidence limit of Reproduction EC10: 29 - 85 mg/L
95% confidence limit of Reproduciton EC50: 76 - 96 mg/L

Reported statistics and error estimates:

Statistical Analysis
ECx Calculations
The 21-Day ECx values for parental generation immobilization were calculated by Linear Interpolation and were determined using the ToxCalc software package (TOXCALC).
The 21-Day ECx values for reproduction and daphnia length were calculated by Probit analysis using Linear Maximum-Likelihood regression and were determined using the ToxRatPro software package (TOXRAT).

NOEC and LOEC Determinations
For the estimation of the LOEC and NOEC values for parental generation immobilization, the number of immobilized adults over the duration of the test for the control and each test group were analyzed using Fisher’s Exact Test using the ToxCalc software package (TOXCALC).
For the estimation of the LOEC and the NOEC values for reproduction, the numbers of live young produced per adult over the duration of the test for the control and each test group were compared using Williams Multiple Sequential t-test incorporating Shapiro-Wilk's test on normal distribution and Levene’s test on variance homogeneity using the ToxRatPro software package (TOXRAT).
Results from the control and each test group’s daphnia length data, determined for the surviving daphnids on termination of the test, were also compared using Williams Multiple Sequential t-test incorporating Shapiro-Wilk's test on normal distribution and Levene’s test on variance homogeneity using the ToxRatPro software package (TOXRAT).

Maximum Acceptable Toxicant Concentration (MATC)
The MATC was calculated using the geometric mean method as follows:
MATC value = √(LOEC x NOEC)
Where:
NOEC = No Observed Effect Concentration (mg/L)
LOEC = Lowest Observed Effect Concentration (mg/L)

Summary of Findings Following the Exposure of Daphnia magna for 21 Days

Nominal Concentration (mg/L)	Percentage Survival of Parental (P1) Generation	Total Number of Live Young	Number of Live Offspring per Parent Surviving to the End of the Test	Number of Live Offspring at the End of the Test per Parent at the Start of the Test Excluding Parental Accidental or Inadvertent Mortalities
Control	100	1041	115.7*	115.7
10	90	1272	136.8	136.8
18	100	1426	142.6	142.6
56	100	1196	119.6	119.6
100	22**	370	68.0	41.1

* Results from 9 replicates. Brood from replicate 5 lost on day 13 prior to counting, therefore data from replicate removed from subsequent analysis

** Replicate 9 was an accidental mortality, therefore, concentration based on 9 replicates

Statistical Analysis

Analysis of Numbers of Live Young Produced per Adult

 

Results from the control and the each test group were comparedwere compared using Williams Multiple Sequential t-test incorporating Shapiro-Wilk's test on normal distribution and Levene’s test on variance homogeneity.

 

Mean and standard deviation values derived from the number of live young produced per surviving adult in the control and each test group were used for this analysis and are given in the table below:

Nominal Concentration (mg/L)		Number of Live Young Produced per Adult by Day 21	Percentage reduction
Control	Mean	115.7	-
	Standard Deviation	8.9
10	Mean	136.8	-18
	Standard Deviation	7.8
18	Mean	142.6	-23
	Standard Deviation	13.5
32	Mean	138.6	-20
	Standard Deviation	9.1
56	Mean	119.6	-3.4
	Standard Deviation	13.7
100	Mean	41.1	65
	Standard Deviation	22.2

  No significant differences (P³0.05) were found between the control and 10, 18, 32 and 56 mg/L testgroups in terms of the number of live young produced per adult by Day 21 using the above methods of statistical analysis,however there was a significant difference between the control and the 100 mg/L test group.

 

Analysis of theDaphniaLength Data

 Results from the control and the each test groupDaphnialength data, determined for the surviving daphnids on termination of the testwere comparedwere compared using Williams Multiple Sequential t-test incorporating Shapiro-Wilk's test on normal distribution and Levene’s test on variance homogeneity.

 

Mean and standard deviation values derived from the length of the surviving daphnids of the parental (P₁) generation in the control and each test group were used for this analysis and are given in the table below:

Nominal Concentration (mg/L)		DaphniaLengths at Day 21 (mm)	Percentage reduction
Control	Mean	4.1	-
	Standard Deviation	0.10
10	Mean	4.1	-0.3
	Standard Deviation	0.07
18	Mean	4.0	1.2
	Standard Deviation	0.07
32	Mean	4.0	3.4
	Standard Deviation	0.10
56	Mean	3.9	4.6
	Standard Deviation	0.08
100	Mean	3.6	13
	Standard Deviation	0.07

No statistically significant differences (P³0.05) were found between the control and the 10 and 18 mg/L test groups in terms of length of the surviving parental daphnids on Day 21 of the test, using the above methods of statistical analyses,however there was a significant difference between the control and the 32, 56 and 100 mg/L test groups.

 

Given that the percentage reduction in body length at 32 and 56 mg/L were less than 5% and therefore below the calculated EC₁₀of 82 mg/L, it was considered that the observed reductions in body length although statistically significant were not necessarily biologically significant. The NOEC and LOEC for body length after 21 days are therefore considered to be 56 mg/L and 100 mg/L respectively by inspection of the data.

 

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Daphnia magna to the test item resulted in significant mortalities at the concentration of 100 mg/L resulting in 78% mortality by Day 21.
The 21-Day EC50 (immobilization) value, based on nominal test concentrations, for the parental daphnia generation (P1) was calculated to be 84 mg/L.
The 21-Day EC50 (reproduction) based on nominal test concentrations was 94 mg/L with 95% confidence limits of 76 to 96 mg/L.
The "Lowest Observed Effect Concentration" (LOEC) and the "No Observed Effect Concentration" (NOEC) for reproduction based on nominal test concentrations were 100 and 56 mg/L respectively.
The "Maximum Acceptable Toxicant Concentration" (MATC) was calculated to be 75 mg/L.

Executive summary:

Long-term toxicity to Daphnia. The chronic toxicity of the test item was assessed in a Daphnia magna reproduction test under semi-static conditions according to 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.

Based on the results of a preliminary range-finding test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to solutions of the test item at nominal concentrations of 10, 18, 32, 56 and 100 mg/L in Elendt M7 media for a period of 21 days.

Analysis of the fresh and old test preparations on days 0, 2, 5, 7, 9, 12, 14, 16, 19 and 21 showed measured test concentrations were all near nominal therefore the test results are based on nominal test concentrations only.

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.

Exposure of Daphnia magna to the test item gave the following results based on the nominal test concentrations:

Endpoint	Concentration (mg/L)
Immobilization EC10	60*
Immobilization EC50	84*
Reproduction EC10	75
95% confidence limits	29 - 85
Reproduction EC50	94
95% confidence limits	76 - 96
NOEC for Reproduction	56
LOEC for Reproduction	100
MATC (Maximum Acceptable Toxicant Concentration)	75

 * Not possible to determine 95% confidence limits as data did not fit available models

Description of key information

Exposure of Daphnia magna to the test item resulted in significant mortalities at the concentration of 100 mg/L resulting in 78% mortality by Day 21.

- The 21-Day EC50 (immobilization) value, based on nominal test concentrations, for the parental daphnia generation (P1) was calculated to be 84 mg/L.

- The 21-Day EC50 (reproduction) based on nominal test concentrations was 94 mg/L with 95% confidence limits of 76 to 96 mg/L.

- The "Lowest Observed Effect Concentration" (LOEC).

- The "No Observed Effect Concentration" (NOEC) for reproduction based on nominal test concentrations were 100 and 56 mg/L respectively.

- The "Maximum Acceptable Toxicant Concentration" (MATC) was calculated to be 75 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

56 mg/L