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Ecotoxicological information

Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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:
no
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:
no
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: 

























































































Day0257912141619
Nominal concentration [mg/L]         
1.567.9 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 7.98.0
6.25 7.9 7.9 7.9 7.9 7.9 7.9 7.9 7.98.0
12.5 7.9 7.9 7.9 7.9 7.9 7.9 7.9 7.98.0
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.:













































































Day0 (a)2 (b)9 (a)12 (b)19 (a)21 (b)
Nominal concentration [mg/L]      
08.79.18.98.38.78.5
1.568.79.28.88.48.78.4
3.138.69.28.98.38.68.3
6.258.79.28.98.48.68.4
12.58.69.18.98.38.78.4
258.69.28.98.28.78.5

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


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













































































Day0 (a)2 (b)9 (a)12 (b)19 (a)21 (b)
Nominal concentration [mg/L]      
07.98.37.97.88.07.8
1.567.98.37.97.88.07.7
3.137.98.37.97.88.07.7
6.257.98.37.87.88.07.7
12.57.98.37.87.87.97.7
257.98.37.87.87.97.7

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:













































































Day0 (a)2 (b)9 (a)12 (b)19 (a)21 (b)
Nominal concentration [mg/L]      
0202020202020
1.56202020202020
3.13202020202020
6.25202020202020
12.5202020202020
25202020202020

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]:













































































Day0 (a)2 (b)9 (a)12 (b)19 (a)21 (b)
Nominal concentration [mg/L]      
02.462.462.452.462.412.44
1.562.462.462.452.462.432.45
3.132.462.462.462.462.422.44
6.252.462.462.462.462.442.46
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/LRep. 1Rep. 2Rep. 3Rep. 4Rep. 5Rep. 6Rep. 7Rep. 8Rep. 9Rep. 10summeansd[%]
01671451591251421691591161711751528152.820.213.2
1.561451581501401641631681591701771594159.411.67.26
3.131741641241691761781911801611541671167.118.411.0
6.251681671631721731681841641631821704170.47.474.38
12.51591531581551541611561781541831611161.110.66.57
25165183157136150160176155T1541436159.613.98.74

 


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/LRep. 1Rep. 2Rep. 3Rep. 4Rep. 5Rep. 6Rep. 7Rep. 8Rep. 9Rep. 10summeansd[%]
000000000000.00.0000.0000.000
1.5600000000000.00.0000.0000.000
3.1300000000000.00.0000.0000.000
6.2500000000000.00.0000.0000.000
12.500001000001.00.1000.316316
2500000000T11.00.1110.333300













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/LRep.1Rep.2Rep.3Rep.4Rep.5Rep.6Rep.7Rep.8Rep.9Rep.10summeansd[%]
02301002110101.001.05105
1.560123010021101.001.05105
3.13121112001090.9000.73882.0
6.25210000001260.6000.843141
12.5101100102280.8000.78998.6
2513101212T1121.330.86665.0













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 concentrationday of the test                     % Mortality (21d)
mg/L0123456789101112131415161718192021 
0101010101010101010101010101010101010101010100%
1.56101010101010101010101010101010101010101010100%
3.13101010101010101010101010101010101010101010100%
6.25101010101010101010101010101010101010101010100%
12.5101010101010101010101010101010101010101010100%
251010101010101010101010101099999999910%

 


Length of the parent animals































































































































    length [mm]   
nominal concentration [mg/L]Rep. 1Rep. 2Rep. 3Rep. 4Rep. 5Rep. 6Rep. 7Rep. 8Rep. 9Rep. 10meansdcv [%] 
04.6194.3814.6674.4764.5244.4764.2864.2864.6194.5244.4860.1342.99
1.564.4294.5244.5714.5244.5714.4764.3334.6674.2384.6194.4950.1312.92
3.134.5714.2864.4294.6674.5714.6194.6674.6674.4294.4294.5330.1322.92
6.254.6674.5244.5714.6194.5244.6674.6674.5244.4294.5244.5710.0811.77
12.54.5714.3334.5244.6674.5714.4764.5244.4294.6194.6674.5380.1052.32
254.4764.5714.4764.3814.4764.2864.4764.476T4.4294.4500.0791.78













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









































































































































































































































































































































































































































































































































































































































































































































































































































































































 day0123456789101112131415161718192021 sum
vessel No.                         
 Ytotal00000000060033004100420043 165
 Ydead0000000000000000000000 0
1Yalive00000000060033004100420043 165
 Saborted0000000001000000000000 1
 Palive1111111111111111111111  
                          
 Ytotal000000000800290142900480055 183
 Ydead0000000000000000000000 0
2Yalive000000000800290142900480055 183
 Saborted0000000001001000000001 3
 Palive1111111111111111111111  
                          
 Ytotal00000000050028004000420042 157
 Ydead0000000000000000000000 0
3Yalive00000000050028004000420042 157
 Saborted0000000000000000001000 1
 Palive1111111111111111111111  
                          
 Ytotal00000000001600340040004330 136
 Ydead0000000000000000000000 0
4Yalive00000000001600340040004330 136
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal00000000060230029000440048 150
 Ydead0000000000000000000000 0
5Yalive00000000060230029000440048 150
 Saborted0000000000000000000001 1
 Palive1111111111111111111111  





































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































 day0123456789101112131415161718192021 sum
vessel No.                         
 Ytotal00000000060250038004200490 160
 Ydead0000000000000000000000 0
6Yalive00000000060250038004200490 160
 Saborted0000000000000010000010 2
 Palive1111111111111111111111  
                          
 Ytotal000000000100031003900460050 176
 Ydead0000000000000000000000 0
7Yalive000000000100031003900460050 176
 Saborted0000000000000000001000 1
 Palive1111111111111111111111  
                          
 Ytotal00000000060210004100440043 155
 Ydead0000000000000000000000 0
8Yalive00000000060210004100440043 155
 Saborted0000000000000000001001 2
 Palive1111111111111111111111  
                          
 Ytotal0000000007009000000000 16
 Ydead0000000000002000000000 2
9Yalive0000000007007000000000 14
 Saborted0000000000000000000000 0
 Palive1111111111111000000000  
                          
 Ytotal00000000050023003500420050 155
 Ydead0000000001000000000000 1
10Yalive00000000040023003500420050 154
 Saborted0000000000000000000001 1
 Palive1111111111111111111111  
                          







summary

Ytotal000000000591669153348122540423084352331 1453
 Ydead0000000001002000000000 3
 Yalive000000000581669151348122540423084352331 1450
 Saborted0000000002001010003014 12
 Palive10101010101010101010101010999999999  
 P = Parent animal, S = Subitane egg, Y = Young                 

 


 


nominal concentration of the test substance: 12.5 mg/L










































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































 day0123456789101112131415161718192021 sum
vessel No.                         
 Ytotal000000000110280039003600045 159
 Ydead0000000000000000000000 0
1Yalive000000000110280039003600045 159
 Saborted0000000000000000000001 1
 Palive1111111111111111111111  
                          
 Ytotal00000000010002801920001812046 153
 Ydead0000000000000000000000 0
2Yalive00000000010002801920001812046 153
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000000140190039004600400 158
 Ydead0000000000000000000000 0
3Yalive000000000140190039004600400 158
 Saborted0000000000000000010000 1
 Palive1111111111111111111111  
                          
 Ytotal00000000070190036004200051 155
 Ydead0000000000000000000000 0
4Yalive00000000070190036004200051 155
 Saborted0000000000000000000001 1
 Palive1111111111111111111111  
                          
 Ytotal00000000050230041004000397 155
 Ydead0000000001000000000000 1
5Yalive00000000040230041004000397 154
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
                          
 Ytotal000000000100180041000410051 161
 Ydead0000000000000000000000 0
6Yalive000000000100180041000410051 161
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000000140180040003800046 156
 Ydead0000000000000000000000 0
7Yalive000000000140180040003800046 156
 Saborted0000000000000000010000 1
 Palive1111111111111111111111  
                          
 Ytotal000000000130220041005000520 178
 Ydead0000000000000000000000 0
8Yalive000000000130220041005000520 178
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal00000000700150046004000460 154
 Ydead0000000000000000000000 0
9Yalive00000000700150046004000460 154
 Saborted0000000000000010000010 2
 Palive1111111111111111111111  
                          
 Ytotal000000000110280034900490052 183
 Ydead0000000000000000000000 0
10Yalive000000000110280034900490052 183
 Saborted0000000000000000001001 2
 Palive1111111111111111111111  
                          







Summary

Ytotal00000000795019028037629029210812177298 1612
 Ydead0000000001000000000000 1
 Yalive00000000794019028037629029210812177298 1611
 Saborted0000000000000010021013 8
 Palive10101010101010101010101010101010101010101010  
 P = Parent animal, S = Subitane egg, Y = Young                 

 


 


nominal concentration of the test substance: 6.25 mg/L














































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































 day0123456789101112131415161718192021 sum
vessel No.                         
 Ytotal000000000110250040000450047 168
 Ydead0000000000000000000000 0
1Yalive000000000110250040000450047 168
 Saborted0000000001000000001000 2
 Palive1111111111111111111111  
                          
 Ytotal000000000150020003700440051 167
 Ydead0000000000000000000000 0
2Yalive000000000150020003700440051 167
 Saborted0000000000000000000001 1
 Palive1111111111111111111111  
                          
 Ytotal000000000140200040004000490 163
 Ydead0000000000000000000000 0
3Yalive000000000140200040004000490 163
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000000180260004100420045 172
 Ydead0000000000000000000000 0
4Yalive000000000180260004100420045 172
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000000160270041004200470 173
 Ydead0000000000000000000000 0
5Yalive000000000160270041004200470 173
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000000150029003800440042 168
 Ydead0000000000000000000000 0
6Yalive000000000150029003800440042 168
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal0000000001803200460026160046 184
 Ydead0000000000000000000000 0
7Yalive0000000001803200460026160046 184
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000000140019038500430045 164
 Ydead0000000000000000000000 0
8Yalive000000000140019038500430045 164
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000000150025003800370048 163
 Ydead0000000000000000000000 0
9Yalive000000000150025003800370048 163
 Saborted0000000000000000000001 1
 Palive1111111111111111111111  
                          
 Ytotal000000000170292003900450050 182
 Ydead0000000000000000000000 0
10Yalive000000000170292003900450050 182
 Saborted0000000000000001000001 2
 Palive1111111111111111111111  
                          







Summary

Ytotal00000000015301599502051980108316096374 1704
 Ydead0000000000000000000000 0
 Yalive00000000015301599502051980108316096374 1704
 Saborted0000000001000001001003 6
 Palive10101010101010101010101010101010101010101010  
 P = Parent animal, S = Subitane egg, Y = Young                 

 


nominal concentration of the test substance: 3.13 mg/L














































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































 day0123456789101112131415161718192021 sum
vessel No.                         
 Ytotal000000001600280045004000450 174
 Ydead0000000000000000000000 0
1Yalive000000001600280045004000450 174
 Saborted0000000000000000000010 1
 Palive1111111111111111111111  
                          
 Ytotal000000001600260040003700045 164
 Ydead0000000000000000000000 0
2Yalive000000001600260040003700045 164
 Saborted0000000000000010000001 2
 Palive1111111111111111111111  
                          
 Ytotal0000000000703300040041300 124
 Ydead0000000000000000000000 0
3Yalive0000000000703300040041300 124
 Saborted0000000000000000001000 1
 Palive1111111111111111111111  
                          
 Ytotal000000000120027003900410050 169
 Ydead0000000000000000000000 0
4Yalive000000000120027003900410050 169
 Saborted0000000000000000001000 1
 Palive1111111111111111111111  
                          
 Ytotal000000000170230045004500460 176
 Ydead0000000000000000000000 0
5Yalive000000000170230045004500460 176
 Saborted0000000000000000000010 1
 Palive1111111111111111111111  
                          
 Ytotal000000001500310044004500430 178
 Ydead0000000000000000000000 0
6Yalive000000001500310044004500430 178
 Saborted0000000000000010000010 2
 Palive1111111111111111111111  
                          
 Ytotal000000001700310047004500510 191
 Ydead0000000000000000000000 0
7Yalive000000001700310047004500510 191
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000000180290043004400046 180
 Ydead0000000000000000000000 0
8Yalive000000000180290043004400046 180
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal0000000001202200231700420045 161
 Ydead0000000000000000000000 0
9Yalive0000000001202200231700420045 161
 Saborted0000000000000000000001 1
 Palive1111111111111111111111  
                          
 Ytotal000000000120024003300390046 154
 Ydead0000000000000000000000 0
10Yalive000000000120024003300390046 154
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          







Summary

Ytotal0000000064717190543028789402561633185232 1671
 Ydead0000000000000000000000 0
 Yalive0000000064717190543028789402561633185232 1671
 Saborted0000000000000020002032 9
 Palive10101010101010101010101010101010101010101010  
 P = Parent animal, S = Subitane egg, Y = Young                 

 


nominal concentration of the test substance: 1.56 mg/L














































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































 day0123456789101112131415161718192021 sum
vessel No.                         
 Ytotal000000000120160035003500470 145
 Ydead0000000000000000000000 0
1Yalive000000000120160035003500470 145
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal0000000012301600390036001438 158
 Ydead0000000000000000000000 0
2Yalive0000000012301600390036001438 158
 Saborted0000000000000000000001 1
 Palive1111111111111111111111  
                          
 Ytotal000000000130017103600360047 150
 Ydead0000000000000000000000 0
3Yalive000000000130017103600360047 150
 Saborted0000000000000000001001 2
 Palive1111111111111111111111  
                          
 Ytotal000000000130190033003100440 140
 Ydead0000000000000000000000 0
4Yalive000000000130190033003100440 140
 Saborted0000000000000000020010 3
 Palive1111111111111111111111  
                          
 Ytotal000000000140250037003600052 164
 Ydead0000000000000000000000 0
5Yalive000000000140250037003600052 164
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000000150031003900360042 163
 Ydead0000000000000000000000 0
6Yalive000000000150031003900360042 163
 Saborted0000000000000000001000 1
 Palive1111111111111111111111  
                          
 Ytotal000000000160027003700380050 168
 Ydead0000000000000000000000 0
7Yalive000000000160027003700380050 168
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000000160254033400390038 159
 Ydead0000000000000000000000 0
8Yalive000000000160254033400390038 159
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000000150030003300400052 170
 Ydead0000000000000000000000 0
9Yalive000000000150030003300400052 170
 Saborted0000000000001000000001 2
 Palive1111111111111111111111  
                          
 Ytotal000000001200270044200430049 177
 Ydead0000000000000000000000 0
10Yalive000000001200270044200430049 177
 Saborted0000000000000000000001 1
 Palive1111111111111111111111  
                          







Summary

Ytotal00000000241170128109119118101382320105368 1594
 Ydead0000000000000000000000 0
 Yalive00000000241170128109119118101382320105368 1594
 Saborted0000000000001000022014 10
 Palive10101010101010101010101010101010101010101010  
 P = Parent animal, S = Subitane egg, Y = Young                 

 


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














































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































 day0123456789101112131415161718192021 sum
vessel No.                         
 Ytotal000000000140030003400430046 167
 Ydead0000000000000000000000 0
1Yalive000000000140030003400430046 167
 Saborted0000000001000000001000 2
 Palive1111111111111111111111  
                          
 Ytotal000000000140013038003900410 145
 Ydead0000000000000000000000 0
2Yalive000000000140013038003900410 145
 Saborted0000000000000010000020 3
 Palive1111111111111111111111  
                          
 Ytotal0000000001300220350023180048 159
 Ydead0000000000000000000000 0
3Yalive0000000001300220350023180048 159
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal00000000001400290040004200 125
 Ydead0000000000000000000000 0
4Yalive00000000001400290040004200 125
 Saborted0000000000000100000000 1
 Palive1111111111111111111111  
                          
 Ytotal000000000120240031003500400 142
 Ydead0000000000000000000000 0
5Yalive000000000120240031003500400 142
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000000170241039000370051 169
 Ydead0000000000000000000000 0
6Yalive000000000170241039000370051 169
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000000160160035000460046 159
 Ydead0000000000000000000000 0
7Yalive000000000160160035000460046 159
 Saborted0000000000000010000001 2
 Palive1111111111111111111111  
                          
 Ytotal00000000001500270033004100 116
 Ydead0000000000000000000000 0
8Yalive00000000001500270033004100 116
 Saborted0000000000000000000100 1
 Palive1111111111111111111111  
                          
 Ytotal000000000180027003500400051 171
 Ydead0000000000000000000000 0
9Yalive000000000180027003500400051 171
 Saborted0000000000000000000001 1
 Palive1111111111111111111111  
                          
 Ytotal000000000160031003600410051 175
 Ydead0000000000000000000000 0
10Yalive000000000160031003600410051 175
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          







Summary

Ytotal00000000012029641245617810573972258381293 1528
 Ydead0000000000000000000000 0
 Yalive00000000012029641245617810573972258381293 1528
 Saborted0000000001000120001122 10
 Palive10101010101010101010101010101010101010101010  
 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 periodInitial48h/72h oldTime-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
1.56Day 0-21.561.471.65100106
Day 9-121.78d1.73d
Day 19-211.631.64
3.13Day 0-23.212.783.25103104
Day 9-123.343.56
Day 19-212.883.56
6.3Day 0-26.375.796.210299
Day 9-126.556.53
Day 19-215.416.27
12.5Day 0-21312.212.7100101
Day 9-1212.712.8
Day 19-2112.213.1
25Day 0-2272425100101
Day 9-122425
Day 19-212427
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 date0 (a)2 (b)2 (c)9 (a)12 (b)12 (c)19 (a)21 (b)21 (c)
Test groupNominal concentration [mg/]Sample Identification Number
00 (Control)11121314151617181
11.5621222324252627282
23.1331323334353637383
36.2541424344454647484
412.551525354555657585
52561626364656667686
 Total primary samples666666666
 Total retain samples (d)121212121212121212
(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 namePreparationExpected contentFound content Mean contentMean recovery
  [mg/L][mg/L][mg/L] (1)[%](1)
1 (2)a0<0.5<0.5-
1 (2)b0<0.5
2 (2)a1.561.561.56100
2 (2)b1.561.57
3 (2)a3.133.173.21103
3 (2)b3.133.25
4 (2)a6.256.326.37102
4 (2)b6.256.43
5 (2)a12.513.013104
5 (2)b12.513.1
6 (2)a252727106
6 (2)b2527

(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 namePreparationExpected contentFound content Mean contentMean recovery
  [mg/L][mg/L][mg/L] (1)[%] (1)
11 (2)a0<0.5<0.5-
11 (2)b0<0.5
12 (2)a1.561.481.4794
12 (2)b1.561.47
13 (2)a3.132.572.7889
13 (2)b3.132.98
14 (2)a6.255.735.7993
14 (2)b6.255.85
15 (2)a12.512.212.297
15 (2)b12.512.1
16 (2)a252525100
16 (2)b2525
21 (2)a0<0.5<0.5-
21 (2)b0<0.5
22 (2)a1.561.621.61103
22 (2)b1.561.6
23 (2)a3.132.963.0096
23 (2)b3.133.05
24 (2)a6.255.495.5689
24 (2)b6.255.63
25 (2)a12.511.911.995
25 (2)b12.511.9
26 (2)a25242496
26 (2)b2524
      
(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 dayPreparationExpected contentFound content Mean contentMean recovery
  [mg/L][mg/L][mg/L] (1)[%](1)
31 (2)a0<0.5<0.5-
31 (2)b0<0.5
32 (2)a1.561.871.89121
32 (2)b1.561.91
33 (2)a3.133.343.34107
33 (2)b3.133.33
34 (2)a6.256.546.55105
34 (2)b6.256.56
35 (2)a12.512.812.7101
35 (2)b12.512.6
36 (2)a252525101
36 (2)b2525
      
(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 namePreparationExpected contentFound content Mean contentMean recovery
  [mg/L][mg/L][mg/L] (1)[%] (1)
41 (2)a0<0.5<0.5-
41 (2)b0<0.5
22 (2)a1.561.941.91123
42 (2)b1.561.89
43 (2)a3.133.643.56114
43 (2)b3.133.49
44 (2)a6.256.56.53104
44 (2)b6.256.56
45 (2)a12.512.612.8103
45 (2)b12.513
46 (2)a25252599
46 (2)b2524
51 (2)a0<0.5<0.5-
51 (2)b0<0.5
52 (2)a1.561.341.3385
52 (2)b1.561.32
53 (2)a3.132.992.9795
53 (2)b3.132.94
54 (2)a6.255.785.7893
44 (2)b6.255.79
55 (2)a12.512.412.298
55 (2)b12.512.1
56 (2)a252525100
56 (2)b2525
      
(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 dayPreparationExpected contentFound content Mean contentMean recovery
  [mg/L][mg/L][mg/L] (1)[%](1)
61 (2)a0<0.5<0.5-
61 (2)b0<0.5
62 (2)a1.561.641.63104
62 (2)b1.561.61
63 (2)a3.132.892.8892
63 (2)b3.132.87
64 (2)a6.255.465.4187
64 (2)b6.255.37
65 (2)a12.512.112.297
65 (2)b12.512.2
66 (2)a25242494
66 (2)b2524
      
(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 namePreparationExpected contentFound content Mean contentMean recovery
  [mg/L][mg/L][mg/L] (1)[%] (1)
71 (2)a0<0.5<0.5-
71 (2)b0<0.5
72 (2)a1.561.661.64105
72 (2)b1.561.61
73 (2)a3.133.593.56114
73 (2)b3.133.52
74 (2)a6.256.226.27100
74 (2)b6.256.32
75 (2)a12.51313.1105
75 (2)b12.513.2
76 (2)a252727108
76 (2)b2527
81 (2)a0<0.5<0.5-
81 (2)b0<0.5
82 (2)a1.561.751.74112
82 (2)b1.561.74
83 (2)a3.133.563.60115
83 (2)b3.133.63
84 (2)a6.256.526.55105
84 (2)b6.256.58
85 (2)a12.513.013.2106
85 (2)b12.513.4
86 (2)a252727107
86 (2)b2527
      
(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 dayPreparationExpected contentFound content Mean contentMean recovery
  [mg/L][mg/L][mg/L] (1)[%](1)
32b (2)a1.561.691.72110
32b (2)b1.561.74
32c (2)a1.561.751.72110
32c (2)b1.561.7
42b (3)a1.561.621.61103
42b (3)b1.561.6
42c (3)a1.561.711.68108
42c (3)b1.561.65
      
(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 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 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

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.