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REACH

Disodium [[N,N'-ethylenebis[N-(carboxymethyl)glycinato]](4-)-N,N',O,O',ON,ON']cuprate(2-)

EC number: 237-864-5 | CAS number: 14025-15-1

Life Cycle description
Uses advised against

Ecotoxicological information

Long-term toxicity to aquatic invertebrates

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Administrative data

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:

from 2021-Jun-14 to 2021-Nov-15, with the definitive exposure phase from 2021-Jun-16 to 2021-Jul-07

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:

2012

Deviations:

yes

Remarks:

The temp. variation for the period was up to 2.5°C. The temperature was in a range of ±2°C with exception of approx.12-hours. This deviation does not affect the overall validity since it hast no influence on the toxicity endpoint determined in this study.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name of test substance: EDTA-CuK2
Test substance No.: 18/0169-2
Batch identification: FC-C 11531
CAS No.: 74181-84-3
Purity: 92.0 % (see Certificate of Analysis)
Identity: Confirmed (see Certificate of Analysis)
Homogeneity: given
Storage stability: Expiry date: Jun 2022
The stability under storage conditions over the exposure period was guaranteed by the sponsor, and the sponsor holds this responsibility.

Chemical Name:
Dipotassium [[N,N'-ethylenebis[N-(carboxymethyl)glycinato]](4-)-N,N',O,O',ON,ON']cuprate(2-)
Synonyms:
Cuprate(2-), [[N,N'-1,2-ethanediylbis[N-[(carboxy-.kappa.O)methyl]glycinato-.kappa.N,.kappa.O]](4-)]-, potassium (1:2),(OC-6-21)-; Ethylenediaminetetraacetic acid, copper dipotassium complex;

Appearance
- physical state: solid
- color: blue

Analytical monitoring:

yes

Remarks:

Method of analysis: Mass spectrometry coupled to an HPLC-system

Details on sampling:

The concentration of the test substance was determined in one representative test interval per week in each freshly prepared test solution (test vessels without daphnids) and in the 48-h or 72-h old test solutions before renewal (combined sample from test vessels with daphnids).
Additionally, samples are collected and analyzed from the 48-hour or 72-hour old abiotic replicate (no Daphnia or food algae) per test group.
The samples were taken from the combined test solution from all test group replicates using a glass pipette and were transported to the analytical laboratory in glass ampoules.

For each concentration, two retained samples were taken at each time point. Retained samples are stored at the Laboratory ECO in a freezer (at approx. –20 °C). Analysis of these samples were performed in case of equivocal analytical results with the original samples or after loss of/ damage to original samples after agreement by the Study Director. Retained samples were described by the suffix “b” and “c” in the report. Following finalization of the report, all analytical samples, including retained samples, will be discarded.

Vehicle:

Details on test solutions:

0 (control), 1.56, 3.13, 6.25, 12.5, 25 mg/L as nominal concentrations based on test substance mass without correction for purity were prepared.

The stability of the test substance as a solution in test media and under testing conditions was determined by concentration control analysis.

The stock test solutions were prepared by dissolving test substance in 1 L of test medium and stirring for
approximately 10 min at ambient temperature. The stock solutions were checked visually to ensure that the test substance
was completely dissolved. The actual mass of test substance weighed to prepare each stock solution is given below.

The actual mass of test substance weighed to prepare each stock solution is given below.
The test solutions were visibly colorless and clear after preparation.
Day: 0 2 5 7 9 12 14 16 19
Nominal concentration [mg/L] Test substance (mg)
1.56 1.57 1.55 1.59 1.56 1.59 1.55 1.60 1.55 1.60
3.13 3.13 3.15 3.17 3.17 3.17 3.17 3.12 3.11 3.19
6.25 6.28 6.27 6.30 6.30 6.27 6.29 6.27 6.30 3.24
12.5 12.57 12.53 12.58 12.59 12.51 12.60 12.59 12.55 12.59
25 25.01 25.27 25.17 25.38 25.08 25.07 25.21 25.08 25.27

Reason for the selection of the test concentrations:
According to the test guidelines, at least 5 concentrations in a geometric series with a separation factor ≤3.2 (OECD [Ref. 2]) should be used.
In a preliminary test (experimental conduct in accordance with GLP but without a GLP status), after 14 days exposure to the test substance, no significant effect on reproduction was
observed at 10 mg/L.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test species : Daphnia magna STRAUS

Reason for selection of the test species:
Recommended species in the test guidelines [Refs. 1–4].

Origin :
The clone of Daphnia magna STRAUS 1820 used was supplied by the Institut National de Recherche Chimique Appliquée,
France, in 1978.
From this date on this clone was cultured and bred continuously in the Ecotoxicology Laboratory of Experimental Toxicology and Ecology, BASF SE, Ludwigshafen Germany.

Culture conditions:
Daphnia brood stock are kept in mass cultures consisting of approx. 30 individuals for a maximum of 4 weeks. All individuals
in the mass culture originate from a single female. After approximately 14 days the adults have produced at least 3 broods and the young can be used in tests. Offspring are
removed from the mass cultures at least once daily during the normal work week to ensure that young daphnia are <24-h old (first instar) at start of exposure. Detailed records are kept (in test facility archives) to monitor the health of Daphnia brood stock cultures including observations of young production, mortality, ephippia, and measurement of water chemistry parameters. Only young from healthy cultures without signs of
stress are used for testing.

Acclimatization:
The Daphnia are cultured under the identical conditions as the test including test media (Elendt M4), water quality, temperature
(20 ±1°C), and diet.

Age at start of exposure: <24 h

Test type:

semi-static

Water media type:

freshwater

Remarks:

Elendt M4, according to OECD 211, Annex 2 is used A synthetic fresh water (Elendt M4) is used as media for culture and test purposes. For the composition of this M4 medium see OECD 211.

Limit test:

Total exposure duration:

21 d

Hardness:

Hardness was measured in the freshly prepared test solution in an additional replicate of each concentration for one interval per week and in the old test solution from the combined replicates of each test group for one interval per week.

Test temperature:

19.5-22.0°C
The recommended test temperature in the test guideline is 18–22°C (max. temperature difference 2°C).
The temperature variation for the period mentioned was up to 2.5°C. The temperature was in a range of ±2°C with exception of approx.12-hours.
The slightly higher measured temperature had no influence on the outcome of this experiment. There was no mortality
or any observed clinical signs of toxicity in this treatment. This deviation does not affect the overall validity since it has no influence
on the toxicity endpoint determined in this study.
For details see section "Any other information on results incl. tables" below.

pH:

For details see section "Any other information on results incl. tables" below.

Dissolved oxygen:

The dissolved oxygen concentration was above 3 mg/L.
For details see section "Any other information on results incl. tables" below.

Nominal and measured concentrations:

0 (control), 1.56, 3.13, 6.25, 12.5, 25 mg/L as nominal concentrations based on test substance mass without correction for purity were prepared.

The test solutions were visibly homogeneous.

Method of analysis: Mass spectrometry coupled to an HPLC-system.

Limit of quantification (LOQ): 0.09 mg/L.

Recovery efficiency: 85-116 %.

All measured concentrations deviated less than 20% from the nominal concentration.
Thus, the test substance was stable in solution under test conditions and nominal concentrations
are an accurate representation of exposure levels maintained throughout the test period. In the 2nd interval the measured concentration of the test substance in the lowest test concentrations
were in a range of 121-123% of the nominal concentration. Therefore, the retain samples were measured and used for the calculation of this concentration. The measured concentrations of
the retained samples were in a range of 103-108% of the nominal concentration (see analytical
report in the appendix). Therefore, the mean value of primary and retained samples were used for calculation.
Following recommendations in OECD 211 the results should be evaluated based on the nominal concentrations.
Additionally, samples were analyzed from the abiotic replicate of each test concentration and the control at the end of the renewal interval. All abiotic replicates were within ±20% of the nominal concentrations. These samples were analyzed to assist in the interpretation of
results in the event that food algae or Daphnia over the exposure period interfered with the test substance analyses. Since the biotic test replicate samples were all within ±20% of the nominal, the additional analyses of abiotic replicates have no influence on the interpretation of this study.

Details on test conditions:

At start of exposure 1 neonate (<24 h old) Daphnia magna was distributed impartially into each of the 10 replicate test vessels per test group. The test vessels were maintained and daphnids fed as described in chapter 3.3. Parent mortality, abnormal effects, and numbers of live and dead offspring were assessed every day throughout the experiment. Reproductive success was measured by counting and discarding the offspring produced by each parent for the duration of the study. Separating neonates from adults was accomplished by gently removing adult daphnids from each chamber by means of a wide bore pipet and pouring remaining water and young daphnids through a fine mesh screen. The young collected on the screen were placed in a petri plate and counted on a light table before being discarded.

On non-renewal days, the old test solution was collected in a clean beaker and returned to the test vessel along with the parent daphnid. On renewal days, the old test solution was collected from all test group replicates. On these days the parent daphnids were returned to their test vessels with fresh test solution.
On day 21 of exposure, the surviving adults were removed from the test vessels and euthanized by immersion in 70% isopropanol. Individuals were then isolated on a labeled glass slide. The body length of each adult daphnid was measured from the apex of the helmet to the base of the posterior spine (i.e. excluding the anal spine and protruding second antennae) with a binocular dissecting microscope (Leitz Diavert) and a calibrated eyepiece micrometer which was checked with a standardized slide.

Throughout the test, the appearance of the test solutions and dissolution behavior of the test
substance was observed and recorded daily. The chemical and physical parameters of the test medium (total hardness, acid capacity, pH, conductivity and total organic carbon) were determined after aeration and prior to use in the test and were within acceptable ranges. Dissolved oxygen, pH and temperature were measured in the old and in the freshly prepared test solution in replicate 1 of each test group for one interval per week. In addition, temperature was measured continuously during the whole exposure period in a separate vessel filled with water proximal to the test vessels. Hardness was measured in the freshly prepared test solution in an additional replicate of each concentration for one interval per week and in the old test solution from the combined replicates of each test group for one interval per week.
The stability of the test substance as a solution in test media and under testing conditions was determined by concentration control analysis. The test solutions were visibly homogeneous.

Acclimatization:
The Daphnia are cultured under the identical conditions as the test including test media (Elendt M4), water quality, temperature (20 ±1°C), and diet.

Age at start of exposure: <24 h

Aeration: none

Test solution renewal: Semi-static, renewal 3 times per week

Diet:
During the test daphnids were fed daily a diet of live green algae Desmodesmus subspicatus,
cultured in a synthetic medium. The algae were separated from their culture medium by
centrifugation, resuspended in daphnid's medium (M4) corresponding to concentrations of 1490 mg and 1500 mg TOC*/L (respectively) in the algal concentrates used.
The daphnids were fed a defined volume (≤134 μL) of the concentrate to reach the amount of food defined in the table below. The algae were stored in a refrigerator (dark, about 4-8°C) for maximum 21 days.
By adding the algal concentrate, the test solution was slightly diluted. During a 72-h test interval with 3 feedings, 0.134 mL each, a total of 0.402 mL are added to 50 mL test volume resulting in a maximal dilution of 0.8 %.

Feeding schedule:

Day of Test Amount of food per parent animal and day [mg TOC] *
0-3 0.10
4-5 0.12
6-7 0.14
8-test end 0.20
*amount of feed algae determined as total organic carbon.

Light intensity / Photo period:
743-799 lux at a wavelength of 400–750 nm. The light intensity did not exceed 1000–1500 lux for cool white light (equivalent to 15–20 μE•m-2•s-1 according to the test guideline.
16 hours light : 8 hours darkness

Test vessels :
Numbered glass beakers (nominal volume 100 mL), covered with glass Petri plates to slow evaporation.

Test volume: 50 mL

Biological loading: 1 animal/test vessel (0.02 animals/mL)

Test groups :
0 (control), 1.56, 3.13, 6.25, 12.5, 25 mg/L as nominal concentrations based on test substance mass without correction for purity.

Replicates/test group:
10 replicates (1 parent animal each)
1 additional abiotic replicate (no Daphnia or food algae) per test group is used to help assess test substance stability over the renewal period.

Reason for the selection of the test concentrations:
According to the test guidelines, at least 5 concentrations in a geometric series with a separation factor ≤3.2 (OECD) should be used.
In a preliminary test (experimental conduct in accordance with GLP but without a GLP status), after 14 days exposure to the test substance, no significant effect on reproduction was observed at 10 mg/L.

Each test group initially consisted of 10 replicates containing one juvenile Daphnia (<24 h old) each.
One additional abiotic replicate (no Daphnia or food algae) per test group was used to help assess test substance stability over the renewal period.
Test group Nominal Concentration as test substance(a)
[mg/L]
0 (Control) 0
1 1.56
2 3.13
3 6.25
4 12.5
5 25
(a): Nominal concentrations based on test substance mass without correction for purity

Test substance preparation:
The stock test solutions were prepared by dissolving test substance in 1 L of test medium and stirring for approximately 10 min at ambient temperature. The stock solutions were checked visually to ensure that the test substance was completely dissolved.

Data evaluation:
The following parameters and statistical procedures (hypothesis tests) were used to evaluate significant differences to the control:
- Reproduction as number of living young (Wilcoxon’s test, one sided with Bonferroni Holm adjustment, one sided analysis)
- Parent mortality as immobilized organisms (Fishers exact test, one sided analysis)

The following additional endpoints undergo statistical evaluation but are not used to establish LOEC and NOEC:
- % immobile young (Wilcoxon’s test, with Bonferroni Holm adjustment, one sided analysis)
- % aborted eggs (Wilcoxon’s test, one sided with Bonferroni Holm adjustment, one sided analysis) - Time to first brood (Wilcoxon’s test, with Bonferroni Holm adjustment, one sided analysis)
- Growth as length in mm (Dunnett´s test, one sided analysis)

The NOEC and LOEC are based on the primary population relevant parameters identified in the OECD test guideline, reproduction and mortality.
For the evaluation of reproduction as number of living young two analyses were done. The first analysis only uses the data of surviving parents.
The second analysis includes the data from all animals which do not die accidentally or inadvertently during the test.
This endpoint is called number of living young per introduced parent.
Any test replicates with documented accidental parent mortality (non-substance related mortality with a known cause) are excluded from the statistical evaluations. To identify inadvertent mortality, a step-down procedure using the Cochran-Armitage trend test,
was applied to the remaining parent mortality data to determine the presence of a concentration response pattern.
The first step uses data from all test groups. When a significant result occurs an additional test was performed omitting the data from the highest test group as the second step etc. This procedure was performed until no significant result occurs. Mortality in test groups with a significant result is considered test substance related. Any mortality in those test groups
without a significant Cochran-Armitage trend test, is considered inadvertent (not substance related with unknown cause) and those replicates are excluded from further analyses. Mortality in the control group of ≤20% is considered inadvertent.
For the analysis of the parent mortality only the accidental deaths with known causes are excluded. The results of the Cochran-Armitage trend test are not used to set NOEC and LOEC values for mortality.
The percent reduction of number of living young, the percent reduction of length and percent
increase in time to first brood were calculated in relation to the control mean. Percentages lower than 0 were set to 0.
The percent immobile young in each test group was calculated in relation to the sum of the number of immobile and number of living young. The percent aborted eggs in each test group was calculated in relation to the sum of number of aborted eggs, number of immobile and number of living young. The endpoints length, time to first brood, immobile young and aborted eggs were evaluated only for organisms that survived to the end of the test.

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 25

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: Active ingredient = 92%

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

Dissolution behavior:
All test solutions were visibly colorless and clear throughout each renewal period.
No undissolved test substance or other unusual manifestations were observed.

Analytical Results:
All measured concentrations deviated less than 20% from the nominal concentration. Thus, the test substance was stable in solution under test conditions and nominal concentrations are an accurate representation of exposure levels maintained throughout the test period. In the 2nd
interval the measured concentration of the test substance in the lowest test concentrations
were in a range of 121-123% of the nominal concentration. Therefore, the retain samples were
measured and used for the calculation of this concentration. The measured concentrations of
the retained samples were in a range of 103-108% of the nominal concentration (see analytical
report in the appendix). Therefore, the mean value of primary and retained samples were used for calculation.
Following recommendations in OECD 211 the results should be evaluated based on the nominal concentrations.
Additionally, samples were analyzed from the abiotic replicate of each test concentration and the control at the end of the renewal interval.
All abiotic replicates were within ±20% of the nominal concentrations (see analytical report in the appendix).
These samples were analyzed to assist in the interpretation of results in the event that food algae or Daphnia over the exposure period interfered with the test substance analyses. Since the biotic test replicate samples were all within ±20% of the nominal, the additional analyses of abiotic replicates have no influence on the interpretation of this study and are only presented in the appendix with the analytical report.

Biological results:
The OECD 211 test guideline is primarily designed to assess effects on Daphnia reproductive output (living young) as the primary population relevant endpoint. Measuring and reporting other biological parameters such as growth, %immobile young, time to first brood and %aborted eggs is optional. These biological parameters in addition to reproduction may assist in the overall interpretation of the study results; however, they alone are not indicative of a population relevant effect. A statistically significant difference in these additional parameters without an effect on reproduction is not considered in establishing the NOEC.

No significant mortality or any other additional significant adverse effects or abnormal behavior
were observed in any of the test treatments. The following overall effect concentrations (mg/L) are based on reproduction (living offspring per surviving adult) as the primary population relevant response and were obtained based on nominal concentrations:
LOEC = > 25 mg/L
NOEC = ≥ 25 mg/L
Since the measured concentrations of the test substance in the test solutions were within +20% of the nominal concentrations, the effect concentration can be expressed relative to the nominal concentration.

In a 21-day chronic toxicity study, Daphnia magna were exposed to EDTA-CuK2 to nominal concentrations of 0 (control) and 1.56, 3.13, 6.25, 12.5, and 25 mg/L under semi-static
conditions in accordance with the OECD 211 guideline. The water pH, temperature and dissolved oxygen were within acceptable guideline specifications. Mortality, reproduction and sublethal effects were monitored daily.
The following overall effect concentrations (mg/L) are based on reproduction (living offspring per surviving adult) as the primary population relevant response and were obtained based on nominal concentrations:
LOEC: > 25 mg/L
NOEC: ≥ 25 mg/L

The data were not sufficient to calculate ECx values for reproduction or mortality.

No significant mortality or any other additional significant adverse effects or abnormal behavior
were observed in any of the test treatments.

The toxicity results presented here are consistent with the results from preliminary tests.

The recommended test temperature in the test guideline is 18–22°C (max. temperature difference 2°C). The temperature variation for the period mentioned was up to 2.5°C. The temperature was in a range of ±2°C with exception of approx. 12-hours. The slightly higher measured temperature had no influence on the outcome of this experiment. There was no mortality or any observed clinical signs of toxicity in this treatment. This deviation does not affect the overall validity since it has no influence on the toxicity endpoint determined in this study.

The results in this study are consistent with all validity criteria and the test is valid according to the guidelines of this study.
No deviations from test guidelines or other incidents occurred during the course of the reported test which may have influenced the results.

Validity criteria:
This test was fully compliant with all the following validity criteria required by the corresponding test guidelines and is considered valid.
• In the control the mortality of parent animals up to the end of the test should be ≤20%. The mortality in the control was 0%
• In the control the mean number of living young per parent animal that survived the test should be ≥60.
In the control the mean number of living young per parent animal was 152.8.

Results with reference substance (positive control):

In order to verify that the Daphnia magna culture is responding normally to toxic stress, acute toxicity tests with a reference substance are conducted monthly. Reference substance tests are conducted generally according to the OECD 202 guideline and in accordance with GLP, but without a GLP status. The EC50(48 h) of the reference substance sodium chloride (NaCl) was 5.43 g/L (experiment date: 26 May 2021, project number: 50E0789/12E064).
This result is within the range of 3.88–7.22 g/L, which represents ±2 standard deviations from the published EC50(48 h) of 5.55 g/L and indicates that the culture of Daphnia magna used in this study is responding normally to toxic stress.

Reported statistics and error estimates:

Tables and/or figures of measured parameters were produced using relevant computing software. All reported data values are rounded to the appropriate significant figures based on the precision of the analytical method and/or consistent with the requirements in the pertinent test guideline(s). T

Calculations to determine the LOEC and NOEC were carried out using SAS System computer software.
The following parameters and statistical procedures (hypothesis tests) were
used to evaluate significant differences to the control:
- Reproduction as number of living young (Wilcoxon’s test, one sided with Bonferroni Holm adjustment, one sided analysis)
- Parent mortality as immobilized organisms (Fishers exact test, one sided analysis)
The following additional endpoints undergo statistical evaluation but are not used to establish LOEC and NOEC:
- % immobile young (Wilcoxon’s test, with Bonferroni Holm adjustment, one sided analysis)
- % aborted eggs (Wilcoxon’s test, one sided with Bonferroni Holm adjustment, one sided analysis)
- Time to first brood (Wilcoxon’s test, with Bonferroni Holm adjustment, one sided analysis)
- Growth as length in mm (Dunnett´s test, one sided analysis)

The NOEC and LOEC are based on the primary population relevant parameters identified in the OECD test guideline, reproduction and mortality. For the evaluation of reproduction
as number of living young two analyses were done. The first analysis only uses the data of
surviving parents. The second analysis includes the data from all animals which do not die
accidentally or inadvertently during the test. This endpoint is called number of living young per
introduced parent.
The percent reduction of number of living young, the percent reduction of length and percent
increase in time to first brood were calculated in relation to the control mean. Percentages
lower than 0 were set to 0.

pH values of each stock solutions:

Day	0	2	5	7	9	12	14	16	19
Nominal concentration [mg/L]
1.56	7.9	7.8	7.9	7.9	7.9	7.9	7.9	7.9	8.0
3.13	7.9	7.9	7.9	7.9	7.9	7.9	7.9	7.9	8.0
6.25	7.9	7.9	7.9	7.9	7.9	7.9	7.9	7.9	8.0
12.5	7.9	7.9	7.9	7.9	7.9	7.9	7.9	7.9	8.0
25	7.9	7.9	7.9	7.9	7.9	7.9	7.9	7.9	7.9

Measured values for the oxygen content [mg/L], measured in replicate 1 per test conc.:

Day	0 (a)	2 (b)	9 (a)	12 (b)	19 (a)	21 (b)
Nominal concentration [mg/L]
0	8.7	9.1	8.9	8.3	8.7	8.5
1.56	8.7	9.2	8.8	8.4	8.7	8.4
3.13	8.6	9.2	8.9	8.3	8.6	8.3
6.25	8.7	9.2	8.9	8.4	8.6	8.4
12.5	8.6	9.1	8.9	8.3	8.7	8.4
25	8.6	9.2	8.9	8.2	8.7	8.5

a: in the freshly prepared test solutions
b: before renewal in the old test solutions

pH-values, measured in replicate 1 per test concentration:

Day	0 (a)	2 (b)	9 (a)	12 (b)	19 (a)	21 (b)
Nominal concentration [mg/L]
0	7.9	8.3	7.9	7.8	8.0	7.8
1.56	7.9	8.3	7.9	7.8	8.0	7.7
3.13	7.9	8.3	7.9	7.8	8.0	7.7
6.25	7.9	8.3	7.8	7.8	8.0	7.7
12.5	7.9	8.3	7.8	7.8	7.9	7.7
25	7.9	8.3	7.8	7.8	7.9	7.7

a: in the freshly prepared test solutions
b: before renewal in the old test solutions

Instantaneous temperature (ºC), measured in replicate 1 per test concentration:

Day	0 (a)	2 (b)	9 (a)	12 (b)	19 (a)	21 (b)
Nominal concentration [mg/L]
0	20	20	20	20	20	20
1.56	20	20	20	20	20	20
3.13	20	20	20	20	20	20
6.25	20	20	20	20	20	20
12.5	20	20	20	20	20	20
25	20	20	20	20	20	20

a: in the freshly prepared test solutions
b: before renewal in the old test solutions

Additionally, temperature was measured continuously during the exposure period in a separate water filled vessel proximal to the test vessels. The temperature ranged from 19.5-22.0°C.
The recommended test temperature in the test guideline is 18–22°C (max. temperature difference 2°C). The temperature variation for the period mentioned was up to 2.5°C. The temperature was in a range of ±2°C with exception of approx. 12-hours. The slightly higher measured temperature had no influence on the outcome of this experiment. There was no mortality or any observed clinical signs of toxicity in this treatment. This deviation does not affect the overall validity since it has no influence on the toxicity endpoint determined in this
study.

Measured values for the total hardness [mmol/L]:

Day	0 (a)	2 (b)	9 (a)	12 (b)	19 (a)	21 (b)
Nominal concentration [mg/L]
0	2.46	2.46	2.45	2.46	2.41	2.44
1.56	2.46	2.46	2.45	2.46	2.43	2.45
3.13	2.46	2.46	2.46	2.46	2.42	2.44
6.25	2.46	2.46	2.46	2.46	2.44	2.46
12.5	- [1]	- [1]	- [1]	- [1]	- [1]	- [1]
25	- [1]	- [1]	- [1]	- [1]	- [1]	- [1]

a: in the freshly prepared test solutions
b: before renewal in the old test solutions

[1]: The determination of water hardness is suitable for determining the amount of metal ions in a solution. Due to the presence of further metal ions, side reactions can take place that may prevent the reaction. Thus, the indicator is blocked, and no color change occurs during the titration. For the concentration 12.5 and 25 mg/L no titration is possible. Since the total hardness in all measured groups was on a same level and no effects on reproductivity or abnormal behavior were observed, it has no influence on the outcome of this study.

Total number of living young per surviving animal after the 21 day exposure period. Base for the data are the values standardized on the number of surviving parent animals of each of the ten replicates.

nominal concentration														cv
mg/L	Rep. 1	Rep. 2	Rep. 3	Rep. 4	Rep. 5	Rep. 6	Rep. 7	Rep. 8	Rep. 9	Rep. 10	sum	mean	sd	[%]
0	167	145	159	125	142	169	159	116	171	175	1528	152.8	20.2	13.2
1.56	145	158	150	140	164	163	168	159	170	177	1594	159.4	11.6	7.26
3.13	174	164	124	169	176	178	191	180	161	154	1671	167.1	18.4	11.0
6.25	168	167	163	172	173	168	184	164	163	182	1704	170.4	7.47	4.38
12.5	159	153	158	155	154	161	156	178	154	183	1611	161.1	10.6	6.57
25	165	183	157	136	150	160	176	155	T	154	1436	159.6	13.9	8.74

Total number of immobile young per surviving parent animal after the 21 day exposure period. Base for the data are the values standardized on the number of surviving parent animals of each of the ten replicates.

nominal concentration														cv
mg/L	Rep. 1	Rep. 2	Rep. 3	Rep. 4	Rep. 5	Rep. 6	Rep. 7	Rep. 8	Rep. 9	Rep. 10	sum	mean	sd	[%]
0	0	0	0	0	0	0	0	0	0	0	0.0	0.000	0.000	0.000
1.56	0	0	0	0	0	0	0	0	0	0	0.0	0.000	0.000	0.000
3.13	0	0	0	0	0	0	0	0	0	0	0.0	0.000	0.000	0.000
6.25	0	0	0	0	0	0	0	0	0	0	0.0	0.000	0.000	0.000
12.5	0	0	0	0	1	0	0	0	0	0	1.0	0.100	0.316	316
25	0	0	0	0	0	0	0	0	T	1	1.0	0.111	0.333	300

T = Parent animal died during the test period

cv = coefficient of variance

sd = standard deviation

Total number of aborted subitane eggs per surviving parent animal after the 21 day exposure period. Base for the data are the values standardized on the number of surviving parent animals of each of the ten replicates.

nominal concentration														cv
mg/L	Rep.1	Rep.2	Rep.3	Rep.4	Rep.5	Rep.6	Rep.7	Rep.8	Rep.9	Rep.10	sum	mean	sd	[%]
0	2	3	0	1	0	0	2	1	1	0	10	1.00	1.05	105
1.56	0	1	2	3	0	1	0	0	2	1	10	1.00	1.05	105
3.13	1	2	1	1	1	2	0	0	1	0	9	0.900	0.738	82.0
6.25	2	1	0	0	0	0	0	0	1	2	6	0.600	0.843	141
12.5	1	0	1	1	0	0	1	0	2	2	8	0.800	0.789	98.6
25	1	3	1	0	1	2	1	2	T	1	12	1.33	0.866	65.0

T = Parent animal died during the test period

cv = coefficient of variance

sd = standard deviation

Total number of surviving parent animals per test day at each concentration

nominal concentration	day of the test																						% Mortality (21d)
mg/L	0	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
0	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	0%
1.56	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	0%
3.13	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	0%
6.25	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	0%
12.5	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	0%
25	10	10	10	10	10	10	10	10	10	10	10	10	10	9	9	9	9	9	9	9	9	9	10%

Length of the parent animals

				length [mm]
nominal concentration [mg/L]	Rep. 1	Rep. 2	Rep. 3	Rep. 4	Rep. 5	Rep. 6	Rep. 7	Rep. 8	Rep. 9	Rep. 10	mean	sd	cv [%]
0	4.619	4.381	4.667	4.476	4.524	4.476	4.286	4.286	4.619	4.524	4.486	0.134	2.99
1.56	4.429	4.524	4.571	4.524	4.571	4.476	4.333	4.667	4.238	4.619	4.495	0.131	2.92
3.13	4.571	4.286	4.429	4.667	4.571	4.619	4.667	4.667	4.429	4.429	4.533	0.132	2.92
6.25	4.667	4.524	4.571	4.619	4.524	4.667	4.667	4.524	4.429	4.524	4.571	0.081	1.77
12.5	4.571	4.333	4.524	4.667	4.571	4.476	4.524	4.429	4.619	4.667	4.538	0.105	2.32
25	4.476	4.571	4.476	4.381	4.476	4.286	4.476	4.476	T	4.429	4.450	0.079	1.78

cv = coefficient of variance

sd = standard deviation

T = Parent animal died during the test period

Table and summarized evaluation of the daily single values of the amount of young (total, alive and dead) of the aborted subitane eggs and the living parent animals.

nominal concentration of the test substance: 25 mg/L

	day	0	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	sum
vessel No.
	Y_total	0	0	0	0	0	0	0	0	0	6	0	0	33	0	0	41	0	0	42	0	0	43	165
	Y_dead	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
1	Y_alive	0	0	0	0	0	0	0	0	0	6	0	0	33	0	0	41	0	0	42	0	0	43	165
	S_aborted	0	0	0	0	0	0	0	0	0	1	0	0	0	0	0	0	0	0	0	0	0	0	1
	P_alive	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1

	Y_total	0	0	0	0	0	0	0	0	0	8	0	0	29	0	14	29	0	0	48	0	0	55	183
	Y_dead	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
2	Y_alive	0	0	0	0	0	0	0	0	0	8	0	0	29	0	14	29	0	0	48	0	0	55	183
	S_aborted	0	0	0	0	0	0	0	0	0	1	0	0	1	0	0	0	0	0	0	0	0	1	3
	P_alive	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1

	Y_total	0	0	0	0	0	0	0	0	0	5	0	0	28	0	0	40	0	0	42	0	0	42	157
	Y_dead	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
3	Y_alive	0	0	0	0	0	0	0	0	0	5	0	0	28	0	0	40	0	0	42	0	0	42	157
	S_aborted	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	1	0	0	0	1
	P_alive	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1

	Y_total	0	0	0	0	0	0	0	0	0	0	16	0	0	34	0	0	40	0	0	43	3	0	136
	Y_dead	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
4	Y_alive	0	0	0	0	0	0	0	0	0	0	16	0	0	34	0	0	40	0	0	43	3	0	136
	S_aborted	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	P_alive	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1

	Y_total	0	0	0	0	0	0	0	0	0	6	0	23	0	0	29	0	0	0	44	0	0	48	150
	Y_dead	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
5	Y_alive	0	0	0	0	0	0	0	0	0	6	0	23	0	0	29	0	0	0	44	0	0	48	150
	S_aborted	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	1	1
	P_alive	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1

day

sum

vessel No.

Y_total

160

Y_dead

Y_alive

160

S_aborted

P_alive

Y_total

176

Y_dead

Y_alive

176

S_aborted

P_alive

Y_total

155

Y_dead

Y_alive

155

S_aborted

P_alive

Y_total

Y_dead

Y_alive

S_aborted

P_alive

Y_total

155

Y_dead

Y_alive

154

S_aborted

P_alive

summary

Y_total

153

225

308

331

1453

Y_dead

Y_alive

151

225

308

331

1450

S_aborted

P_alive

P = Parent animal, S = Subitane egg, Y = Young

nominal concentration of the test substance: 12.5 mg/L

day

sum

vessel No.

Y_total

159

Y_dead

Y_alive

159

S_aborted

P_alive

Y_total

153

Y_dead

Y_alive

153

S_aborted

P_alive

Y_total

158

Y_dead

Y_alive

158

S_aborted

P_alive

Y_total

155

Y_dead

Y_alive

155

S_aborted

P_alive

Y_total

155

Y_dead

Y_alive

154

S_aborted

P_alive

Y_total

161

Y_dead

Y_alive

161

S_aborted

P_alive

Y_total

156

Y_dead

Y_alive

156

S_aborted

P_alive

Y_total

178

Y_dead

Y_alive

178

S_aborted

P_alive

Y_total

154

Y_dead

Y_alive

154

S_aborted

P_alive

Y_total

183

Y_dead

Y_alive

183

S_aborted

P_alive

Summary

Y_total

190

376

292

108

177

298

1612

Y_dead

Y_alive

190

376

292

108

177

298

1611

S_aborted

P_alive

P = Parent animal, S = Subitane egg, Y = Young

nominal concentration of the test substance: 6.25 mg/L

day

sum

vessel No.

Y_total

168

Y_dead

Y_alive

168

S_aborted

P_alive

Y_total

167

Y_dead

Y_alive

167

S_aborted

P_alive

Y_total

163

Y_dead

Y_alive

163

S_aborted

P_alive

Y_total

172

Y_dead

Y_alive

172

S_aborted

P_alive

Y_total

173

Y_dead

Y_alive

173

S_aborted

P_alive

Y_total

168

Y_dead

Y_alive

168

S_aborted

P_alive

Y_total

184

Y_dead

Y_alive

184

S_aborted

P_alive

Y_total

164

Y_dead

Y_alive

164

S_aborted

P_alive

Y_total

163

Y_dead

Y_alive

163

S_aborted

P_alive

Y_total

182

Y_dead

Y_alive

182

S_aborted

P_alive

Summary

Y_total

153

159

205

198

108

316

374

1704

Y_dead

Y_alive

153

159

205

198

108

316

374

1704

S_aborted

P_alive

P = Parent animal, S = Subitane egg, Y = Young

nominal concentration of the test substance: 3.13 mg/L

day

sum

vessel No.

Y_total

174

Y_dead

Y_alive

174

S_aborted

P_alive

Y_total

164

Y_dead

Y_alive

164

S_aborted

P_alive

Y_total

124

Y_dead

Y_alive

124

S_aborted

P_alive

Y_total

169

Y_dead

Y_alive

169

S_aborted

P_alive

Y_total

176

Y_dead

Y_alive

176

S_aborted

P_alive

Y_total

178

Y_dead

Y_alive

178

S_aborted

P_alive

Y_total

191

Y_dead

Y_alive

191

S_aborted

P_alive

Y_total

180

Y_dead

Y_alive

180

S_aborted

P_alive

Y_total

161

Y_dead

Y_alive

161

S_aborted

P_alive

Y_total

154

Y_dead

Y_alive

154

S_aborted

P_alive

Summary

Y_total

190

287

256

163

185

232

1671

Y_dead

Y_alive

190

287

256

163

185

232

1671

S_aborted

P_alive

P = Parent animal, S = Subitane egg, Y = Young

nominal concentration of the test substance: 1.56 mg/L

day

sum

vessel No.

Y_total

145

Y_dead

Y_alive

145

S_aborted

P_alive

Y_total

158

Y_dead

Y_alive

158

S_aborted

P_alive

Y_total

150

Y_dead

Y_alive

150

S_aborted

P_alive

Y_total

140

Y_dead

Y_alive

140

S_aborted

P_alive

Y_total

164

Y_dead

Y_alive

164

S_aborted

P_alive

Y_total

163

Y_dead

Y_alive

163

S_aborted

P_alive

Y_total

168

Y_dead

Y_alive

168

S_aborted

P_alive

Y_total

159

Y_dead

Y_alive

159

S_aborted

P_alive

Y_total

170

Y_dead

Y_alive

170

S_aborted

P_alive

Y_total

177

Y_dead

Y_alive

177

S_aborted

P_alive

Summary

Y_total

117

128

109

191

181

138

232

105

368

1594

Y_dead

Y_alive

117

128

109

191

181

138

232

105

368

1594

S_aborted

P_alive

P = Parent animal, S = Subitane egg, Y = Young

nominal concentration of the test substance: 0 mg/L (Control)

day

sum

vessel No.

Y_total

167

Y_dead

Y_alive

167

S_aborted

P_alive

Y_total

145

Y_dead

Y_alive

145

S_aborted

P_alive

Y_total

159

Y_dead

Y_alive

159

S_aborted

P_alive

Y_total

125

Y_dead

Y_alive

125

S_aborted

P_alive

Y_total

142

Y_dead

Y_alive

142

S_aborted

P_alive

Y_total

169

Y_dead

Y_alive

169

S_aborted

P_alive

Y_total

159

Y_dead

Y_alive

159

S_aborted

P_alive

Y_total

116

Y_dead

Y_alive

116

S_aborted

P_alive

Y_total

171

Y_dead

Y_alive

171

S_aborted

P_alive

Y_total

175

Y_dead

Y_alive

175

S_aborted

P_alive

Summary

Y_total

120

124

178

105

225

293

1528

Y_dead

Y_alive

120

124

178

105

225

293

1528

S_aborted

P_alive

P = Parent animal, S = Subitane egg, Y = Young

Summarized analytically measured concentrations of EDTA-CuK2 in the test solutions:

		Analytically Measured Concentration
Nominal Concentration [mg/L]	Renewal period	Initial	48h/72h old	Time-weighted Mean
		[mg/L]	[mg/L]	[mg/L]a	[%]b	[%]c
0 (Control)	Day 0-2	<0.5	<0.5	-	-	-
	Day 9-12	<0.5	<0.5
	Day 19-21	<0.5	<0.5
1.56	Day 0-2	1.56	1.47	1.65	100	106
	Day 9-12	1.78d	1.73d
	Day 19-21	1.63	1.64
3.13	Day 0-2	3.21	2.78	3.25	103	104
	Day 9-12	3.34	3.56
	Day 19-21	2.88	3.56
6.3	Day 0-2	6.37	5.79	6.2	102	99
	Day 9-12	6.55	6.53
	Day 19-21	5.41	6.27
12.5	Day 0-2	13	12.2	12.7	100	101
	Day 9-12	12.7	12.8
	Day 19-21	12.2	13.1
25	Day 0-2	27	24	25	100	101
	Day 9-12	24	25
	Day 19-21	24	27
a: based on 3 renewal period days.
b: % of Mean Initial
c: % of Nominal concentration
d: mean of primary and retained samples

Sampling scheme during the study:

Study date		0 (a)	2 (b)	2 (c)	9 (a)	12 (b)	12 (c)	19 (a)	21 (b)	21 (c)
Test group	Nominal concentration [mg/]	Sample Identification Number
0	0 (Control)	1	11	21	31	41	51	61	71	81
1	1.56	2	12	22	32	42	52	62	72	82
2	3.13	3	13	23	33	43	53	63	73	83
3	6.25	4	14	24	34	44	54	64	74	84
4	12.5	5	15	25	35	45	55	65	75	85
5	25	6	16	26	36	46	56	66	76	86
	Total primary samples	6	6	6	6	6	6	6	6	6
	Total retain samples (d)	12	12	12	12	12	12	12	12	12
(a): Fresh test solution
(b): Aged test solution (48-72 hours old)
(c): Abiotic replicate (no Daphnia or food algae)
(d): Retained samples are indicated with a "b" or "c" in addition to the sample number

Summarized results of the first set of samples:

Sample name	Preparation	Expected content	Found content	Mean content	Mean recovery
		[mg/L]	[mg/L]	[mg/L] (1)	[%](1)
1 (2)	a	0	<0.5	<0.5	-
1 (2)	b	0	<0.5	<0.5	-
2 (2)	a	1.56	1.56	1.56	100
2 (2)	b	1.56	1.57	1.56	100
3 (2)	a	3.13	3.17	3.21	103
3 (2)	b	3.13	3.25	3.21	103
4 (2)	a	6.25	6.32	6.37	102
4 (2)	b	6.25	6.43	6.37	102
5 (2)	a	12.5	13.0	13	104
5 (2)	b	12.5	13.1	13	104
6 (2)	a	25	27	27	106
6 (2)	b	25	27	27	106

(1) Calculation is based on non-rounded values
(2) Sample production: Jun 16, 2021; Sampling on: Jun 16 2021

Summarized results of the second set of samples

Sample name	Preparation	Expected content	Found content	Mean content	Mean recovery
		[mg/L]	[mg/L]	[mg/L] (1)	[%] (1)
11 (2)	a	0	<0.5	<0.5	-
11 (2)	b	0	<0.5	<0.5	-
12 (2)	a	1.56	1.48	1.47	94
12 (2)	b	1.56	1.47	1.47	94
13 (2)	a	3.13	2.57	2.78	89
13 (2)	b	3.13	2.98	2.78	89
14 (2)	a	6.25	5.73	5.79	93
14 (2)	b	6.25	5.85	5.79	93
15 (2)	a	12.5	12.2	12.2	97
15 (2)	b	12.5	12.1	12.2	97
16 (2)	a	25	25	25	100
16 (2)	b	25	25	25	100
21 (2)	a	0	<0.5	<0.5	-
21 (2)	b	0	<0.5	<0.5	-
22 (2)	a	1.56	1.62	1.61	103
22 (2)	b	1.56	1.6	1.61	103
23 (2)	a	3.13	2.96	3.00	96
23 (2)	b	3.13	3.05	3.00	96
24 (2)	a	6.25	5.49	5.56	89
24 (2)	b	6.25	5.63	5.56	89
25 (2)	a	12.5	11.9	11.9	95
25 (2)	b	12.5	11.9	11.9	95
26 (2)	a	25	24	24	96
26 (2)	b	25	24	24	96

(1) Calculation is based on non-rounded values
(2) Sample production: Jun 16, 2021; Sampling on: Jun 16 2021

Summarized results of the third set of samples

Sampling day	Preparation	Expected content	Found content	Mean content	Mean recovery
		[mg/L]	[mg/L]	[mg/L] (1)	[%](1)
31 (2)	a	0	<0.5	<0.5	-
31 (2)	b	0	<0.5	<0.5	-
32 (2)	a	1.56	1.87	1.89	121
32 (2)	b	1.56	1.91	1.89	121
33 (2)	a	3.13	3.34	3.34	107
33 (2)	b	3.13	3.33	3.34	107
34 (2)	a	6.25	6.54	6.55	105
34 (2)	b	6.25	6.56	6.55	105
35 (2)	a	12.5	12.8	12.7	101
35 (2)	b	12.5	12.6	12.7	101
36 (2)	a	25	25	25	101
36 (2)	b	25	25	25	101

(1) Calculation is based on non-rounded values
(2) Sample production: Jun 25, 2021; Sampling on: Jun 25 2021

Summarized results of the fourth set of samples

Sample name	Preparation	Expected content	Found content	Mean content	Mean recovery
		[mg/L]	[mg/L]	[mg/L] (1)	[%] (1)
41 (2)	a	0	<0.5	<0.5	-
41 (2)	b	0	<0.5	<0.5	-
22 (2)	a	1.56	1.94	1.91	123
42 (2)	b	1.56	1.89	1.91	123
43 (2)	a	3.13	3.64	3.56	114
43 (2)	b	3.13	3.49	3.56	114
44 (2)	a	6.25	6.5	6.53	104
44 (2)	b	6.25	6.56	6.53	104
45 (2)	a	12.5	12.6	12.8	103
45 (2)	b	12.5	13	12.8	103
46 (2)	a	25	25	25	99
46 (2)	b	25	24	25	99
51 (2)	a	0	<0.5	<0.5	-
51 (2)	b	0	<0.5	<0.5	-
52 (2)	a	1.56	1.34	1.33	85
52 (2)	b	1.56	1.32	1.33	85
53 (2)	a	3.13	2.99	2.97	95
53 (2)	b	3.13	2.94	2.97	95
54 (2)	a	6.25	5.78	5.78	93
44 (2)	b	6.25	5.79	5.78	93
55 (2)	a	12.5	12.4	12.2	98
55 (2)	b	12.5	12.1	12.2	98
56 (2)	a	25	25	25	100
56 (2)	b	25	25	25	100

(1) Calculation is based on non-rounded values
(2) Sample production: Jun 25, 2021; Sampling on: Jun 28 2021

Summarized results of the fifth set of samples

Sampling day	Preparation	Expected content	Found content	Mean content	Mean recovery
		[mg/L]	[mg/L]	[mg/L] (1)	[%](1)
61 (2)	a	0	<0.5	<0.5	-
61 (2)	b	0	<0.5	<0.5	-
62 (2)	a	1.56	1.64	1.63	104
62 (2)	b	1.56	1.61	1.63	104
63 (2)	a	3.13	2.89	2.88	92
63 (2)	b	3.13	2.87	2.88	92
64 (2)	a	6.25	5.46	5.41	87
64 (2)	b	6.25	5.37	5.41	87
65 (2)	a	12.5	12.1	12.2	97
65 (2)	b	12.5	12.2	12.2	97
66 (2)	a	25	24	24	94
66 (2)	b	25	24	24	94

(1) Calculation is based on non-rounded values
(2) Sample production: Jul 05, 2021; Sampling on: Jul 05 2021

Summarized results of the sixth set of samples

Sample name	Preparation	Expected content	Found content	Mean content	Mean recovery
		[mg/L]	[mg/L]	[mg/L] (1)	[%] (1)
71 (2)	a	0	<0.5	<0.5	-
71 (2)	b	0	<0.5	<0.5	-
72 (2)	a	1.56	1.66	1.64	105
72 (2)	b	1.56	1.61	1.64	105
73 (2)	a	3.13	3.59	3.56	114
73 (2)	b	3.13	3.52	3.56	114
74 (2)	a	6.25	6.22	6.27	100
74 (2)	b	6.25	6.32	6.27	100
75 (2)	a	12.5	13	13.1	105
75 (2)	b	12.5	13.2	13.1	105
76 (2)	a	25	27	27	108
76 (2)	b	25	27	27	108
81 (2)	a	0	<0.5	<0.5	-
81 (2)	b	0	<0.5	<0.5	-
82 (2)	a	1.56	1.75	1.74	112
82 (2)	b	1.56	1.74	1.74	112
83 (2)	a	3.13	3.56	3.60	115
83 (2)	b	3.13	3.63	3.60	115
84 (2)	a	6.25	6.52	6.55	105
84 (2)	b	6.25	6.58	6.55	105
85 (2)	a	12.5	13.0	13.2	106
85 (2)	b	12.5	13.4	13.2	106
86 (2)	a	25	27	27	107
86 (2)	b	25	27	27	107

(1) Calculation is based on non-rounded values
(2) Sample production: Jul 05, 2021; Sampling on: Jul 07 2021

Summarized result of the retained set of samples

Sampling day	Preparation	Expected content	Found content	Mean content	Mean recovery
		[mg/L]	[mg/L]	[mg/L] (1)	[%](1)
32b (2)	a	1.56	1.69	1.72	110
32b (2)	b	1.56	1.74	1.72	110
32c (2)	a	1.56	1.75	1.72	110
32c (2)	b	1.56	1.7	1.72	110
42b (3)	a	1.56	1.62	1.61	103
42b (3)	b	1.56	1.6	1.61	103
42c (3)	a	1.56	1.71	1.68	108
42c (3)	b	1.56	1.65	1.68	108

(1) Calculation is based on non-rounded values
(2) Sample production: Jun 25, 2021; Sampling on: Jul 25 2021
(3) Sample production: Jun 25, 2021; Sampling on: Jul 28, 2021

Validity criteria fulfilled:

yes

Conclusions:

The effects on reproduction were evaluated based on the reproduction (living offspring per surviving adult) as the primary population relevant response for the most sensitive endpoint (reproduction) and were obtained based on nominal concentrations:
LOEC > 25 mg/L
NOEC >= 25 mg/L

Since the analytically determined concentrations of the test substance in the test solutions were within the range of ±20% of the nominal concentrations, the effect concentration can be expressed relative to the nominal concentration.
No significant mortality, reduced reproduction, or any other additional significant adverse effects or abnormal behavior were observed in any of the test treatments. The data were not sufficient to calculate ECx values for reproduction or mortality.

Executive summary:

A Daphnia magna reproduction test (semi-static, 21 d) of the test item EDTA-CuK2 (batch number: FC-C 11531) was conducted according to OECD 211 (2012) from 2021-Jun-08 to 2022-Feb-28, with the definitive exposure phase from 2021-Jun-16 to 2021-Jul-07 at BASF SE.
Test species was Daphnia magna STRAUS. Ten daphnids, held individually, were used per nominal test item concentration and control. At the start of the exposure the daphnids were less than 24 hours old. The study was carried out under semi-static conditions with medium exchange three times per week. Aim of the test was to assess the effects on the reproduction capacity and other test item-related effects or parameters such as the intrinsic rate of the natural increase, the number of broods with living juveniles, first appearance of living juveniles, occurrence of aborted eggs and stillborn juveniles, adult mortality, body length and dry weight of the parental daphnids.

The test item EDTA-CuK2 is a blue solid. The aqueous media were prepared dissolving test substance in 1 L of test medium and stirring for approximately 10 min at ambient temperature. Five test concentrations were prepared and tested nominal concentration rates 25 – 12.5 – 6.25 – 3.13 - 1.56 mg//L (separation factor 2.0).
The stock solutions were checked visually to ensure that the test substance was completely dissolved.
The test item concentrations of EDTA-CuK2 were analytically verified via LC-MS three times during the test (once within a period of 7 days) in the fresh media at the start of an exposure-renewal interval (0 hours; on test days 0, 9 and 19) as well as in the old media at the end of an exposure-renewal interval (24 hours; on test days 2, 12 and 21) in all test item concentrations and in the control.
The environmental conditions were within the acceptable limits. The validity criteria of the test guideline were met.

The OECD 211 test guideline is primarily designed to assess effects on Daphnia reproductive output (living young) as the primary population relevant endpoint. Measuring and reporting other biological parameters such as growth, %immobile young, time to first brood and %aborted eggs is optional. These biological parameters in addition to reproduction may assist in the overall interpretation of the study results; however, they alone are not indicative of a population relevant effect. A statistically significant difference in these additional parameters without an effect on reproduction is not considered in establishing the NOEC. No significant mortality or any other additional significant adverse effects or abnormal behavior were observed in any of the test treatments. The following overall effect concentrations (mg/L) are based on reproduction (living offspring per surviving adult) as the primary population relevant response and were obtained based on nominal concentrations:
LOEC = > 25 mg/L
NOEC = ≥ 25 mg/L
Since the measured concentrations of the test substance in the test solutions were within +20% of the nominal concentrations, the effect concentration can be expressed relative to the nominal concentration.

The nominal concentrations in the report are based on test substance mass without correction for purity. The purity indicated on the CoA is 92%.

The for purity corrected effect concentrations are:

LOEC = > 23 mg/L
NOEC = ≥ 23 mg/L

Description of key information

The results of a long-term daphnia test with source substance EDTA-CuK2 is, after approval by ECHA (colla project), read across to target substance EDTA-CuNa2.

A Daphnia magna reproduction test (semi-static, 21 d) of the test item EDTA-CuK2 was conducted according to OECD 211 (2012) under GLP conditions.
Test species was Daphnia magna STRAUS. Ten daphnids, held individually, were used per nominal test item concentration and control. At the start of the exposure the daphnids were less than 24 hours old. The study was carried out under semi-static conditions with medium exchange three times per week. Aim of the test was to assess the effects on the reproduction capacity and other test item-related effects or parameters such as the intrinsic rate of the natural increase, the number of broods with living juveniles, first appearance of living juveniles, occurrence of aborted eggs and stillborn juveniles, adult mortality, body length and dry weight of the parental daphnids.

The nominal concentrations in the report are based on test substance mass without correction for purity. The purity indicated on the CoA is 92%.

The for purity corrected effect concentrations are:

LOEC = > 23 mg EDTA-CuK2/L or 21.27 mg EDTA-CuNa2
NOEC = ≥ 23 mg EDTA-CuK2/L or 21.27 mg EDTA-CuNa2

MW EDTA-CuK2 = 430.0 g/mol

MW EDTA-CuNa2 = 397.7 g/mol

Key value for chemical safety assessment

Fresh water invertebrates

Dose descriptor:: NOEC
Effect concentration:: 21.27 mg/L

Additional information

In addition to the long-term daphnia test with EDTA-CuK2 which resulted in a NOEC of 23 mg a.i./L there are two reliable 21 d chronic toxicity studies, one with EDTA-Na2H2 and EDTA-MnNa2 for daphnids available, both with comparable results as observed for EDTA-CuK2.

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