Hexasodium 2,2'-[vinylenebis[(3-sulphonato-4,1-phenylene)imino[6-morpholino-1,3,5-triazine-4,2-diyl]imino]]bis(benzene-1,4-disulphonate)

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

other: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

November 20th to December 11th, 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Reliability of original study is 1

Justification for type of information:

A justification for the read across is given in section 13 of IUCLID.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Sampling for the analytical determination
At the start of the exposure intervals on day 0, 9 and 14, samples of the fresh media were taken after preparation of all test item concentrations and analyzed.
At the end of the exposure intervals on day 2, 12 and 16 (48 or 72 hours), samples of the old media were taken directly from the test vessels.
For the longest exposure-renewal interval of 72 hours, samples were taken at the start (0 hours) and at the end of the exposure interval (72 hours) once within the test period.
Since 100 % mortality occurred at the highest concentration level 13.4 mg/l, analyses were carried out on days 0, 2, 9 and 12 only.

Vehicle:

no

Details on test solutions:

A stock solution of 120 mg/l of the test item was freshly prepared with dilution water for each exposure renewal interval. An appropriate volume of the test item was weighed out and transferred into a measuring flask with an appropriate amount of the dilution water. The stock solution was treated with ultrasound for 1 minute at room temperature. The stock solution was used for the preparation of further test concentrations by dilution with the dilution water.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna STRAUS
- Age at study initiation: less than 24 hours old daphnids. juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period were used for the test. No first brood progeny was used for the test
- Method of breeding: obtained from continuous laboratory cultures. Breeder - Noack Laboratorien GmbH, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany
- Source: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany; Culture
- Culture: In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20 ± 2 °C, 16 hours illumination; light intensity of max 1500 lx; Culture medium -Elendt M4, according to OECD 211
- Feeding of the culture stocks: the culture daphnids are fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus. The algae are cultured at the test facility.
- Feeding during test: Daily; Pseudokirchneriella subcapitata (0.524 - 0.814 ml) and Desmodesmus subspicatus (0.381 - 0.575 ml) suspension was provided as food corresponding to 0.2 mg C per Daphnia and day. The density of the algae suspension varied, but it was the same for all test groups on each feeding day.

ACCLIMATION
- Acclimatization was not necessary, because the composition of the dilution water was equivalent to the culture medium.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

261-280 mg/l as CaCO3 (control)
253-268 mg/l as CaCO3 (highest concentration)
The total hardness was above 140 mg/l as CaCO3.

Test temperature:

19.5-20.8 °C

pH:

7.2-7.82 (control)
7.26 - 7.94 (highest concentration level)
Significant deviations ≥ 1.5 units between the initial and final pH-values were not observed.

Dissolved oxygen:

6.37-8.83 mg/l (control)
6.96-9.27 mg/l (highest concentration level)
The dissolved oxygen concentration was above 3 mg/l.

Nominal and measured concentrations:

0.478 - 1.10 - 2.53 - 5.82 - 13.4 mg/l (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers (5 (ID) x 8 (H) cm), 100 ml capacity
- Type: closed; loosely covered with watch glasses
- FIll volume: 50 ml
- Aeration: no
- Renewal rate of test solution: every two or three days (i.e. on Monday, Wednesday and Friday). For this purpose, a second set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred by pipette
- No. of organisms per vessel: 10 daphnids in 10 replicates were used for all concentration levels and the control
- No. of vessels per concentration: 10
- No. of vessels per control: 10 replicates of dilution water without test item
- Biomass loading rate: one daphnid per replicate
- Application: 50 g test solution per replicate were weighed out into each test vessel. This corresponds to 50 mL. The daphnids were transferred in a small amount of dilution water or test solution by pipette.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: same composition as the culture medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: The water quality parameters (i.e. pH-values, dissolved oxygen concentration, total hardness and water temperature) were measured once within 7 days, in fresh media at the start (0 hours) and in old media at the end (48 or 72 hours) of an exposure-renewal interval, in one replicate of the control and the highest test item concentration with a living parent animal. Since 100% mortality were observed at the highest concentration level (13.4 mg/l) on day 12, the measurements were carried out thereafter in the following concentration level (5.82 mg/l) with living parental daphnids for the remaining exposure interval (day 14 and 16). The water quality parameters in fresh media at the start of an exposure-renewal interval were measured in an additional replicate without daphnids of the highest test item concentration and the control. At the end of an exposure-renewal interval, the water quality parameters of the old media were measured in a test vessel of the highest test item concentration and the control, which contained daphnids and food algae. The temperature in the incubator was recorded throughout the test period with a thermo-hygrograph.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16/8 hours light/dark cycle
- Light intensity: Max. 1500 lx

EFFECT PARAMETERS MEASURED:
- Mortality of parental daphnids: The number of parental daphnids, which were immobilized or dying during the test and the dates of death were observed and recorded once a day at least at the same time as the offspring was counted. Dead daphnids were removed as soon as they were detected.
- Neonates: First appearance of juveniles was checked daily. The number of neonates (alive and dead progeny) was counted and dates of release were recorded once a day from the first day of appearance of neonates until the end of the exposure. The neonates (alive and dead) were removed after counting and before addition of algae to prevent them from consuming food intended for the adults. The number of aborted eggs or dead offspring and dates of observation were recorded.
- Condition of parental daphnids: Abnormalities (e.g. abnormal swimming behavior, number of males and winter eggs) were observed and recorded on each day of observation.
- Total body length / Mean dry weight of parental daphnids: At the end of the test, the total length excluding the anal spine of each survived parental daphnid and the mean dry weight of the survived parental daphnids of all test concentrations and the control were determined. As stated in the OECD protocol, this may be useful additional information but is not used for determination of a NOEC.
- Additional endpoints The time to first brood, the intrinsic rate of population increase and the number and size of first brood per animal were reported, but not used for endpoint calculations.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.3
- Justification for using less concentrations than requested by guideline:
- Range finding study: A non-GLP preliminary range finding test was conducted at the test facility as a shortened reproduction test (at least until observation of the first brood in all test groups) under semi-static conditions with daily renewal of the test solutions over a period of 9 days. A control and three test concentrations of the test item of 1.20, 12.0 and 120 mg/l were tested in the range finding test. The preliminary range finding test will be conducted under diffuse light conditions (light intensity of max. 1500 lx, 16/8 hours light/dark cycle). Five daphnids, one daphnia per replicate, were exposed to each concentration level and the control. The test item concentrations were analytically verified via HPLC-DAD in the fresh media at the start of the exposure and in the old media at the end of an exposure-renewal interval
- Test concentrations (main study): 0.478 - 1.10 - 2.53 - 5.82 - 13.4 mg/l

STATISTICAL EVALUATION
- The NOEC and LOEC for the reproduction and the adult mortality were determined with the software ToxRat Professional. Significant differences of reproduction were determined in comparison to the control using statistical standard procedures as normality test (Shapiro-Wilk’s test), variance homogeneity test (Levene’s test), multiple sequentially-rejective median (2x2-Table) test after Bonferroni-Holm for reproduction and step-down Cochran-Armitage test for adult mortality.
Prior to running the median test as well as the step-down Cochran-Armitage test, a monotonicity test, a normality test and a variance homogeneity test were performed. P-values of the normality and the variance homogeneity test were 0.01. The a-value (acceptable probability of incorrectly concluding that there is a difference) was a= 0.01 for the normality test and the variance homogeneity test. For the effects on reproduction, the juveniles per introduced parent at the start of the test excluding accidental and inadvertent mortality (reproductive output) were counted. Since no accidental or inadvertent mortality was observed, no parental daphnids were excluded from the evaluation of the reproductive output. The coefficients of variation around the mean number of living offspring produced per introduced parent and per survived parent in the control and the test groups were evaluated.
- Determination of the EC- and LC-values: The EC10 / 50 -values for the reproductive output with 95 % confidence limits were calculated by point estimates from the 3-parametric normal CDF with the software ToxRat Professional. The LC20- and the LC50-value for the adult mortality with 95 % confidence limits were calculated by probit analysis with the software toxRat Professional. As the software extrapolated the lower confidence limit for the LC20 (which was outside the tested range) the adjacent test concentration was given. The LC100-value for the adult mortality was empirically derived from the observed data.
- Calculation of the intrinsic rate: The intrinsic rate of natural increase (IR) was calculated for the survived parental daphnids with the following EULER-LOTKA-equation with the software. The intrinsic rate of natural increase is a measure of population growth which integrates reproductive output and age-specific mortality. In steady state populations, it will be zero. For growing populations, it will be positive and for shrinking populations, it will be negative. Clearly, the latter is not sustainable and ultimately will lead to extinction.

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

1.94 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% confidence limits: 1.45 – 2.59 mg/L

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

3.07 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% confidence limits: 1.98 – 4.66

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

5.82 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

2.53 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

2.42 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

adult

Remarks on result:

other: confidence limits: 1.39 – 4.19

Duration:

21 d

Dose descriptor:

other: LC20

Effect conc.:

0.889 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

adult mortality

Remarks on result:

other: confidence limits: < 0.478 – 1.52

Duration:

21 d

Dose descriptor:

other: LC100

Effect conc.:

13.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

adult mortality

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

2.53 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

adult mortality

Remarks on result:

other: Overall effect threshold

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

adult mortality

Remarks on result:

other: Overall effect threshold

Details on results:

Reproductive output: The average number of living juveniles at the end of the test after 21 days per survived parental daphnids was 87.7 in the control group and thus the validity criterion for the reproduction of a mean of ≥60 living offspring per survived parent animal in the control was met. No parental daphnids were excluded due to accidental or inadvertent mortality from the evaluation of the reproductive output. Since the Cochran-Armitage test procedure revealed a significant increasing trend (p ≤0.05) in adult mortality, the evaluation was done based on the cumulative offspring per introduced parent.

Adult Mortality: accidental and inadvertent mortality as defined in the OECD test guideline was not observed. The test item induced statistically significant adult mortality of 100 % after 21 days in the concentration level 13.4 mg/l, 80 % in the concentration level 5.82 mg/l and 30 % in the concentration level 2.53 mg/l (Step-down Cochran-Armitage Test Procedure, α = 0.05). In the concentration levels 0.478 and 1.10 mg/l, a mortality rate of 20 % of the introduced adults was observed, which is not statistically significant. In the concentration levels 0.478 and 1.10 mg/l, a mortality rate of 20 % of the introduced adults was observed, which is not statistically significant. In the control, no mortality of the introduced adults was observed until the end of the test (21 days).

First Appearance of living Juveniles: The first appearance of living juveniles was observed between days 7 and 11 at the introduced parental daphnids of the control and the concentration levels 0.478 to 5.82 mg/l. However, several daphnids died before producing juveniles in the two highest concentration levels.

Stillborn Juveniles and Aborted Eggs: In the control as well as in the concentration levels 0.478 to 13.4 mg/l no stillborn juveniles or aborted eggs were observed during the exposure period of 21 days.

Growth (Total Body Length and Mean Dry Weight) of the Survived Parental Daphnids: The mean values of the body length (excluding the anal spine) of the survived parental daphnids in the tested concentration levels 0.478 to 5.82 mg/l were in the range of 2.00 to 4.44 mm per daphnid and 4.44 mm per daphnid in the control group. The mean dry weight of the survived parental daphnids was in the range of 0.06 to 0.29 mg per daphnid in the concentration levels 0.478 to 5.82 mg/l and 0.32 mg/l per daphnid in the control.

Presence of Males: No males were observed in the control or in the test groups during the test.

Occurrence of Ephippia (Winter Eggs): No ephippia were observed in the control or in the test groups during the test.

Measured exposure concentrations: The measured concentrations of the test item in fresh media at the start of the respective exposure renewal intervals (0 hours) were in the range of 88 to 107% of the nominal concentrations. At the end of the respective exposure-renewal intervals (48 and 72 hours), the measured test item concentrations in the old media were in the range of 90 to 104 % of the nominal concentrations.

Water Quality Parameters: The measured water quality parameters (i.e. pH-value, dissolved oxygen concentration, total water hardness and water temperature) were within the acceptable limits.

Metabolite screening: An aged sample of the highest test concentration was measured via LC-Q-TOF (non-GLP method) for confirmation of two isomers of the test item resulting in two chromatogram peaks at retention time approx. 5.6 and 5.9 min. It is noteworthy that on basis of the measured accurate masses, only the molecular formula was predicted. The chemical structure of the compounds have not been determined. Besides these two isomers, no significant metabolites have been identified in aged samples. In comparison to control samples, an additional compound inherent in the test item was found to be present in freshly prepared and aged samples (retention time approx. 8.2 min), presumably resulting from an impurity. This compound was not included in the quantitative analysis.

Reported statistics and error estimates:

Reproductive output: No parental daphnids were excluded due to accidental or inadvertent mortality from the evaluation of the reproductive output. Since the Cochran-Armitage test procedure revealed a significant increasing trend (p ≤0.05) in adult mortality, the evaluation was done based on the cumulative offspring per introduced parent. A statistically significant reduction of the reproductive output in comparison to the reproductive output in the control was determined at the concentration levels 5.82 and 13.4 mg/l (Multiple sequentially-rejective median (2x2-Table) test after Bonferroni-Holm, α = 0.05). The determination of the EC10- and the EC50-value and their 95% confidence limits was carried out by point estimates from the 3-parametric normal CDF.
Adult mortality: The Cochran-Armitage test procedure revealed a significant increasing trend (p ≤ 0.05) in adult mortality. Therefore, the LC20- and LC50-values were calculated.

Effects on Reproduction for all Introduced Parents (based on nominal concentrations of the test item)

Nominal test item concentrations [mg/l]	Mean number of offspring per introduced parental daphnid
	Mean	SD	CV
13.4	0.0	0.00	not applicable
5.82	4.0	5.37	134.4
2.53	70.1	23.90	34.1
1.10	103.1	30.27	29.4
0.478	108.3	31.07	28.7
Control	87.7	9.92	11.3

 

Mortality [%] of the Adult Daphnids after 7, 14 and 21 Days of Exposure (n = 10)

Nominal test item concentrations [mg/l]	Adult mortality (%)
	7 days	14 days	21 days
13.4	70	100	100*
5.82	0	70	80*
2.53	0	10	30*
1.10	0	10	20
0.478	0	10	20
Control	0	0	0

*= statistically significant (Step-down Cochran-Armitage Test Procedure, α = 0.05)

 

Intrinsic Rates of Natural Increase (IR)

Nominal test item concentrations [mg/l]	Mean IR MV±SD	CV (%)
13.4	Not applicable (no offspring, 100 % adult mortality)
5.82	0.138 ± 0.0457	33.2
2.53	0.351 ± 0.0430	12.3
1.10	0.347 ± 0.0195	5.6
0.478	0.357 ± 0.0235	6.6
Control	0.322 ± 0.0125	3.9

 

First Appearance of Living Juveniles in the Individual Groups

Nominal test item concentrations [mg/l]	Day of first appearance of living juveniles at the introduced parental daphnids in replicate no.										First appearance mean day
	1	2	3	4	5	6	7	8	9	10
13.4	-	-	-	-	-	-	-	-	-	-	not applicable
5.82	-	10	-	-	-	10	10	-	10	-	10.0
2.53	9	11	8	10	8	10	7	9	9	10	9.1
1.10	10	9	8	8	8	10	10	10	10	8	9.1
0.478	10	10	10	10	9	8	10	9	10	10	9.6
Control	10	10	10	9	10	10	10	10	10	10	9.9

-- = not applicable, since no living juveniles occurred

 

Total Body Length and Dry Weight of the Survived Parental Daphnids

Nominal test item concentrations [mg/l]	Total length of the survived parent animals [mm] in replicate no.										MV (mm)	N	Dry weight (mg)
	1	2	3	4	5	6	7	8	9	10			Σ	MV
13.4	-	-	-	-	-	-	-	-	-	-	-	0	-	-
5.82	-	-	2.5	-	1.5	-	-	-	-	-	2.00	2	0.2	0.10
2.53	4.0	4.0	4.0	-	4.0	3.5	-	4.0	-	4.0	3.93	7	0.4	0.06
1.10	4.5	4.0	-	4.5	4.5	4.5	-	4.5	4.5	4.5	4.44	8	0.5	0.06
0.478	4.5	4.5	4.5	4.5	4.5	4.5	-	4.5	-	4.0	4.44	8	2.3	0.29
Control	4.5	4.5	4.5	4.0	4.5	4.0	4.5	4.5	4.5	4.5	4.40	10	3.2	0.32

N = number of the survived parental daphnids

-- = not applicable, due to the mortality of the parental daphnid(s)

 

Measured Concentrations of the Test Item during the Definitive Test

Sampling date	Day 0 Fresh medium 0 hours		Day 2 Old medium 48 hours		Day 9 Fresh medium 0 hours		Day 12 Old medium 72 hours		Day 14 Fresh medium, 0 hours		Day 16 Old medium, 48 hours
Nominal test item concentrations [mg/l]
	Meas. conc. [mg/l]	%	Meas. conc. [mg/l]	%	Meas. conc. [mg/l]	%	Meas. conc. [mg/l]	%	Meas. conc. [mg/l]	%	Meas. conc. [mg/l]	%
13.4	12.4	93	13.0	97	14.4	107	13.8	103	Not analyzed due to 100% adult mortalit
5.82	5.31	91	5.58	96	5.99	103	6.05	104	5.96	102	5.72	98
2.53	2.29	90	2.42	96	2.70	107	2.60	103	2.58	102	2.46	97
1.10	0.972	88	0.990	90	1.02	92	0.994	90	1.08	98	1.03	94
0.478	0.446	93	0.459	96	0.507	106	0.479	100	0.453	95	0.461	96
QC	/	99	/	99	/	102	/	120	/	98	/	103
Control	< LOQ		< LOQ		< LOQ		< LOQ		< LOQ		< LOQ

Meas. conc. = measured concentration of the test item, dilution factors taken into account

% = percent of the nominal concentration of the test item

LOQ = limit of quantification (0.300 mg/L of the test item)

QC = quality control

Immobilization and Offspring in the non-GLP Preliminary Range Finding Test after 9 Days (n = 5, divided into 5 replicates with 1 daphnid each)

Nominal test item concentrations [mg/l]	Mortality [%] after 9 days						Number of offspring after 9 days per inserted parent						First appearance of living juveniles
	Replicates						Replicates
	1	2	3	4	5	MV	1	2	3	4	5	MV	(d)
120	100	100	100	100	100	100	0	0	0	0	0	0	No juveniles occurred
12.0	0*	0*	0*	0*	0*	0	0	0	0	0	0	0	No juveniles occurred
1.20	0	0	0	0	0	0	10	9	12	9	14	11	8
Control	0	0	0	0	0	0	11	10	14	11	11	11	8

* = animal was pale

Validity criteria fulfilled:

yes

Remarks:

The actual mortality of the parent animal in the control group was 0 %; Mean number of living offspring produced per survived parent animal at the end of the test was 87.7 in the control group(required:at least 60 juveniles per survived parental daphnid).

Conclusions:

NOEC (21d, overall threshold effects) =1.10 mg/l (nominal)
NOEC (21d, reproduction) = 2.53 mg/l (nominal)

Executive summary:

A reproduction test on Daphnia magna (semi-static, 21 days) was carried out to assess the effect of the test item on the reproductive output of Daphnia magna, according to the OECD Guideline 211.

Ten daphnids, held individually, were used per concentration level and control. Nominal concentrations of the test item were selected based on the results of a preliminary range finding test (non-GLP) as follows: 0.478 - 1.10 - 2.53 - 5.82 - 13.4 mg/l (separation factor 2.3). The effects on the reproduction capacity and other test item-related effects or parameters such as the intrinsic rate of the natural increase, first appearance of living juveniles, occurrence of aborted eggs and stillborn juveniles, adult mortality, body length and dry weight of the parental daphnids, were assessed. The test item concentrations were analytically verified via HPLC-DAD in the fresh media at the start of the exposure-renewal intervals on days 0, 9, 14 (0 hours) and in the old media at the end of the exposure intervals on days 2, 12, 16 (48 or 72 hours, respectively) in all concentration levels (with surviving daphnids) and in the control. Since 100 % mortality occurred at the highest concentration level 13.4 mg/l, analyses were carried out on days 0, 2, 9 and 12 only. The measured concentrations of the test item in fresh media at the start of the respective exposure renewal intervals (0 hours) were in the range of 88 to 107 % of the nominal concentrations. At the end of the respective exposure-renewal intervals (48 and 72 hours), the measured test item concentrations in the old media were in the range of 90 to 104 % of the nominal concentrations.

The environmental conditions were within the acceptable limits. The validity criteria of the test guideline were met. The effects on reproduction were evaluated based on the reproduction per introduced parent animal, since this parameter is the ecologically most relevant response variable and is required by the OECD test guideline 211 (2012), when a significant trend in mortality is detected.

The overall effect threshold for effects of the test item under the test conditions was 1.10 mg/l (NOEC) and 2.53 mg/l (LOEC) based on the nominal concentrations.

Description of key information

NOEC (21d, overall threshold effects) = 1.10 mg/l (nominal)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1.1 mg/L

Additional information

No data is available for the evaluation of the long-term toxicity to aquatic invertebrates of the substance. For this reason, data on similar substance OB 1 -MSA is used. A read across justification is given in section 13 of the IUCLID dossier.

A reproduction test on Daphnia magna (semi-static, 21 days) was carried out to assess the effect of the test item on the reproductive output of Daphnia magna, according to the OECD Guideline 211. Ten daphnids, held individually, were used per concentration level and control. Nominal concentrations of the test item were selected based on the results of a preliminary range finding test (non-GLP) as follows: 0.478 - 1.10 - 2.53 - 5.82 - 13.4 mg/l (separation factor 2.3). The effects on the reproduction capacity and other test item-related effects or parameters such as the intrinsic rate of the natural increase, first appearance of living juveniles, occurrence of aborted eggs and stillborn juveniles, adult mortality, body length and dry weight of the parental daphnids, were assessed. The test item concentrations were analytically verified via HPLC-DAD in the fresh media at the start of the exposure-renewal intervals on days 0, 9, 14 (0 hours) and in the old media at the end of the exposure intervals on days 2, 12, 16 (48 or 72 hours, respectively) in all concentration levels (with surviving daphnids) and in the control. Since 100 % mortality occurred at the highest concentration level 13.4 mg/l, analyses were carried out on days 0, 2, 9 and 12 only. The measured concentrations of the test item in fresh media at the start of the respective exposure renewal intervals (0 hours) were in the range of 88 to 107% of the nominal concentrations. At the end of the respective exposure-renewal intervals (48 and 72 hours), the measured test item concentrations in the old media were in the range of 90 to 104 % of the nominal concentrations.

The environmental conditions were within the acceptable limits. The validity criteria of the test guideline were met. The effects on reproduction were evaluated based on the reproduction per introduced parent animal, since this parameter is the ecologically most relevant response variable and is required by the OECD test guideline 211 (2012), when a significant trend in mortality is detected.

The overall effect threshold for effects of the test item under the test conditions was 1.10 mg/l (NOEC) and 2.53 mg/l (LOEC) based on the nominal concentrations.

The use of the similar substance OB 1 -MSA is due to the fact that the effect levels obtained in the long-term toxicity study to aquatic invertebrates is the lowest among the ones available in the category. Moreover, the degree of sulfonation and the structural similarity is taken into account. Following a conservative approach, the results obtained in the long-term toxicity study on acquatic invertebrates on the similar substance OB 1 -MSA will be used for the chemical safety assessment.

More details on the results of the other substances in the Category will be provided in the attached document under "Ecotoxicological Information".