Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Currently viewing:

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-Jul-07 to 2021--Nov-12, with the definitive exposure phase of the 2nd experiment from 2021-Sep-22 to 2021-Oct-13
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:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Name of test substance: EDTA-ZnK2
Test substance No.: 20/0482-1
Batch identification: DC-C 11530
CAS No.: 14689-29-3
Purity: 90.4 % (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-(carboxylatomethyl) glycinato]] (4-)-N,N',O,O',ON,ON']zincate(2-)
Synonyms: Ethylenediaminetetraacetic acid, Zinc dipotassium complex
Appearance
- physical state: solid
- color: white

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 (25 mg/L) were prepared by dissolving 50 mg of test substance in 2-L of test medium and stirring for approximately 10 min at ambient temperature. The lower test concentrations were prepared by dilution of this stock solution. Before the dilution was performed, the stock solution was checked for complete dissolution of the test substance.


The actual mass of test substance weighed to prepare each stock solution is given below.

Day: 0 2 5 7 9 12 14 16 19
Test substance (mg) 50.80 50.38 50.88 50.06 50.69 49.97 50.17 50.55 50.56


pH values
Day: 0 2 5 7 9 12 14 16 19
0 mg/L 7.9 8.0 7.9 7.8 7.7 7.8 7.8 7.9 7.8
25 mg/L 7.8 7.8 7.8 7.8 7.8 7.8 7.8 7.8 7.8


Dilutions scheme
Nominal conc. [mg/L] Volume of stock solution [mL] Final Volume of test media [mL]
0 (control) - 1000
1.56 63 1000
3.13 125 1000
6.25 250 1000
12.5 500 1000

The test solutions were visibly colorless and clear after preparation.

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 12 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.

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.
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:
19.4-20.3 °C.
The temperature was in a range of ±1°C.
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): 0.1 mg/L.

Recovery efficiency: 87-106 %.

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.
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. 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 1460 mg and 1513 mg TOC*/L (respectively) in the algal concentrates used.
The daphnids were fed a defined volume (≤137 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, 0.137 mL each, a total of 0.411 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:
801.8-837.4 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 12 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 (25 mg/L) were prepared by dissolving 50 mg of test substance in 2-L
of test medium and stirring for approximately 10 min at ambient temperature. The lower test
concentrations were prepared by dilution of this stock solution. Before the dilution was performed, the stock solution was checked for complete dissolution of the test substance.

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)



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. 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.

Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 25
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: Purity 90.4%
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: Purity 90.4%
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:
Recovery effficiency: 87-106%
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. 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)nas 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 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 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 139.7.

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 3.88 g/L (experiment date: 22 Sep 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.

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]      
09.09.18.89.39.08.8
1.568.99.08.89.38.98.8
3.139.39.08.89.18.98.7
6.259.39.18.79.38.98.6
12.59.29.18.89.38.98.8
258.99.18.89.38.98.8

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]      
07.88.07.78.07.87.9
1.567.88.17.77.97.87.8
3.137.88.07.87.87.87.8
6.257.88.07.87.87.87.7
12.57.88.17.87.87.87.7
257.88.17.87.87.87.7

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]      
019.820.219.919.919.919.6
1.5619.820.219.919.819.819.6
3.1319.820.219.919.919.919.7
6.2519.820.119.920.019.919.6
12.519.820.119.919.919.819.7
2519.820.019.919.919.819.6

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 19.4-20.3°C.


 


 


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













































































Day0 (a)2 (b)9 (a)12 (b)19 (a)21 (b)
Nominal concentration [mg/L]      
02.342.372.392.392.352.26
1.562.362.372.402.382.342.30
3.132.362.372.392.382.292.29
6.252.342.372.372.392.322.27
12.52.342.372.372.392.272.28
252.362.372.372.392.282.31

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


Total number of living 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[%]
01581251651511651211391261341131397139.719.013.6
1.56157165142155921721361431261361424142.422.716.0
3.13175176147166143154991641481221494149.424.116.1
6.251571451311831591541541461491481526152.613.38.7
12.51611631611231581561511461541391512151.212.48.2
25153128125144154136115111851551306130.622.617.3













 
cv = coefficient of variance
sd = standard deviation

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[%]
000000000000.00.00.00.0
1.5600000210003.00.30.7225.0
3.1300000000000.00.00.00.0
6.2500000000000.00.00.00.0
12.500000000000.00.00.00.0
2500000000000.00.00.00.0













 
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[%]
0001010001030.30.5161.0
1.56001010100030.30.5161.0
3.13000000000000.00.00.0
6.25000000100010.10.3316.2
12.5100011110050.50.5105.4
25000000200020.20.6316.2













 
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%
25101010101010101010101010101010101010101010100%

 


Length of the parent animals































































































































    length [mm]   
nominal concentration [mg/L]Rep. 1Rep. 2Rep. 3Rep. 4Rep. 5Rep. 6Rep. 7Rep. 8Rep. 9Rep. 10meansdcv [%] 
04.0484.4764.4294.6674.3334.5244.4764.4294.4294.2864.4100.1643.71
1.564.6194.5714.5244.5244.5714.5244.2864.5714.1904.4764.4860.1383.08
3.134.6194.4294.4764.2864.3814.5244.1434.6194.2384.3334.4050.1593.61
6.254.3814.4294.3814.6194.3334.4764.5714.3334.5244.5244.4570.1012.26
12.54.5714.4294.4294.4294.3814.5244.4763.8104.2864.4764.3810.2154.91
254.1904.5244.4294.5244.4764.4294.2864.2864.4764.3814.4000.1132.56










cv = coefficient of variance
sd = standard deviation

 














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.                         
 Ytotal000000000100270032000410043 153
 Ydead0000000000000000000000 0
1Yalive000000000100270032000410043 153
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal00000000001800310039103900 128
 Ydead0000000000000000000000 0
2Yalive00000000001800310039103900 128
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal00000000001600350003400400 125
 Ydead0000000000000000000000 0
3Yalive00000000001600350003400400 125
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000000140024003100360039 144
 Ydead0000000000000000000000 0
4Yalive000000000140024003100360039 144
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000000135027003700320040 154
 Ydead0000000000000000000000 0
5Yalive000000000135027003700320040 154
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  













































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































 Ytotal000000000110019003200360038 136
 Ydead0000000000000000000000 0
6Yalive000000000110019003200360038 136
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal00000000000160026003600037 115
 Ydead0000000000000000000000 0
7Yalive00000000000160026003600037 115
 Saborted0000000000010000010000 2
 Palive1111111111111111111111  
                          
 Ytotal00000000015002400360036000 111
 Ydead0000000000000000000000 0
8Yalive00000000015002400360036000 111
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal0000000000140030004100000 85
 Ydead0000000000000000000000 0
9Yalive0000000000140030004100000 85
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000000140026004000370038 155
 Ydead0000000000000000000000 0
10Yalive000000000140026004000370038 155
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          







Summary

Ytotal000000000775343120965817680712183940235 1306
 Ydead0000000000000000000000 0
 Yalive000000000775343120965817680712183940235 1306
 Saborted0000000000010000010000 2
 Palive10101010101010101010101010101010101010101010  
 P = Parent animal, S = Subitane egg, Y = Young                 

 














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: 12.5 mg/L








































































































































































































































































































































































































































































































































































































































































































































































































































































































 day0123456789101112131415161718192021 sum
vessel No.                         
 Ytotal000000000130280003700440039 161
 Ydead0000000000000000000000 0
1Yalive000000000130280003700440039 161
 Saborted0000000000000000001000 1
 Palive1111111111111111111111  
                          
 Ytotal000000000120290038000410043 163
 Ydead0000000000000000000000 0
2Yalive000000000120290038000410043 163
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000000130028003800390043 161
 Ydead0000000000000000000000 0
3Yalive000000000130028003800390043 161
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal00000000001800320049000240 123
 Ydead0000000000000000000000 0
4Yalive00000000001800320049000240 123
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000001400310036000410036 158
 Ydead0000000000000000000000 0
5Yalive000000001400310036000410036 158
 Saborted0000000000010000000000 1
 Palive1111111111111111111111  













































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































 Ytotal000000000160033003900360032 156
 Ydead0000000000000000000000 0
6Yalive000000000160033003900360032 156
 Saborted0000000000000000001000 1
 Palive1111111111111111111111  
                          
 Ytotal000000000100025003800400038 151
 Ydead0000000000000000000000 0
7Yalive000000000100025003800400038 151
 Saborted0000000000000000001000 1
 Palive1111111111111111111111  
                          
 Ytotal000000000130025003800380032 146
 Ydead0000000000000000000000 0
8Yalive000000000130025003800380032 146
 Saborted0000000000000000001000 1
 Palive1111111111111111111111  
                          
 Ytotal00000000080030003600370043 154
 Ydead0000000000000000000000 0
9Yalive00000000080030003600370043 154
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000000120012003800380039 139
 Ydead0000000000000000000000 0
10Yalive000000000120012003800380039 139
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          







Summary

Ytotal00000000149718881533274264490354024345 1512
 Ydead0000000000000000000000 0
 Yalive00000000149718881533274264490354024345 1512
 Saborted0000000000010000004000 5
 Palive10101010101010101010101010101010101010101010  
 P = Parent animal, S = Subitane egg, Y = Young                 

 














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: 6.25 mg/L








































































































































































































































































































































































































































































































































































































































































































































































































































































































 day0123456789101112131415161718192021 sum
vessel No.                         
 Ytotal0000000011002900380025150039 157
 Ydead0000000000000000000000 0
1Yalive0000000011002900380025150039 157
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000000150027003200340037 145
 Ydead0000000000000000000000 0
2Yalive000000000150027003200340037 145
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal00000000001700300041004300 131
 Ydead0000000000000000000000 0
3Yalive00000000001700300041004300 131
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000001520350046000430042 183
 Ydead0000000000000000000000 0
4Yalive000000001520350046000430042 183
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000000140024004100420038 159
 Ydead0000000000000000000000 0
5Yalive000000000140024004100420038 159
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  













































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































 Ytotal000000000120250029000450043 154
 Ydead0000000000000000000000 0
6Yalive000000000120250029000450043 154
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal0000000001502400132500390038 154
 Ydead0000000000000000000000 0
7Yalive0000000001502400132500390038 154
 Saborted0000000000000000001000 1
 Palive1111111111111111111111  
                          
 Ytotal000000000110021003700410036 146
 Ydead0000000000000000000000 0
8Yalive000000000110021003700410036 146
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000000120026003500360040 149
 Ydead0000000000000000000000 0
9Yalive000000000120026003500360040 149
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000000120300037000370032 148
 Ydead0000000000000000000000 0
10Yalive000000000120300037000370032 148
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          







Summary

Ytotal0000000026931714398301631704125332430345 1526
 Ydead0000000000000000000000 0
 Yalive0000000026931714398301631704125332430345 1526
 Saborted0000000000000000001000 1
 Palive10101010101010101010101010101010101010101010  
 P = Parent animal, S = Subitane egg, Y = Young                 

 














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: 3.13 mg/L








































































































































































































































































































































































































































































































































































































































































































































































































































































































 day0123456789101112131415161718192021 sum
vessel No.                         
 Ytotal000000001400320044003900046 175
 Ydead0000000000000000000000 0
1Yalive000000001400320044003900046 175
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal0000000016100220046004100041 176
 Ydead0000000000000000000000 0
2Yalive0000000016100220046004100041 176
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000000130028003800340034 147
 Ydead0000000000000000000000 0
3Yalive000000000130028003800340034 147
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000000120310042000400041 166
 Ydead0000000000000000000000 0
4Yalive000000000120310042000400041 166
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal00000000070029003200370038 143
 Ydead0000000000000000000000 0
5Yalive00000000070029003200370038 143
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  













































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































 Ytotal000000000150290033000400037 154
 Ydead0000000000000000000000 0
6Yalive000000000150290033000400037 154
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal00000000001100220031103400 99
 Ydead0000000000000000000000 0
7Yalive00000000001100220031103400 99
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000000110290039000420043 164
 Ydead0000000000000000000000 0
8Yalive000000000110290039000420043 164
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000000130022003400360043 148
 Ydead0000000000000000000000 0
9Yalive000000000130022003400360043 148
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000000170023003200380012 122
 Ydead0000000000000000000000 0
10Yalive000000000170023003200380012 122
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          







Summary

Ytotal00000000309811143102222041363181267340335 1494
 Ydead0000000000000000000000 0
 Yalive00000000309811143102222041363181267340335 1494
 Saborted0000000000000000000000 0
 Palive10101010101010101010101010101010101010101010  
 P = Parent animal, S = Subitane egg, Y = Young                 

 














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: 1.56 mg/L








































































































































































































































































































































































































































































































































































































































































































































































































































































































 day0123456789101112131415161718192021 sum
vessel No.                         
 Ytotal000000001400300034003900400 157
 Ydead0000000000000000000000 0
1Yalive000000001400300034003900400 157
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000001500310039004000040 165
 Ydead0000000000000000000000 0
2Yalive000000001500310039004000040 165
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000000150028003400360029 142
 Ydead0000000000000000000000 0
3Yalive000000000150028003400360029 142
 Saborted0000000000000000001000 1
 Palive1111111111111111111111  
                          
 Ytotal000000001400230042003500041 155
 Ydead0000000000000000000000 0
4Yalive000000001400230042003500041 155
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000001400800200016180016 92
 Ydead0000000000000000000000 0
5Yalive000000001400800200016180016 92
 Saborted0000000000000000001000 1
 Palive1111111111111111111111  













































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































 Ytotal000000001700350042004000040 174
 Ydead0000000020000000000000 2
6Yalive000000001500350042004000040 172
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000000100025003300370032 137
 Ydead0000000000000001000000 1
7Yalive000000000100025003200370032 136
 Saborted0000000000000000001000 1
 Palive1111111111111111111111  
                          
 Ytotal000000000110022003200370041 143
 Ydead0000000000000000000000 0
8Yalive000000000110022003200370041 143
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal00000000001800290038004100 126
 Ydead0000000000000000000000 0
9Yalive00000000001800290038004100 126
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal00000000001900280043100450 136
 Ydead0000000000000000000000 0
10Yalive00000000001900280043100450 136
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          







Summary

Ytotal00000000743637127755717799811711284185239 1427
 Ydead0000000020000001000000 3
 Yalive00000000723637127755717798811711284185239 1424
 Saborted0000000000000000003000 3
 Palive10101010101010101010101010101010101010101010  
 P = Parent animal, S = Subitane egg, Y = Young                 

 














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: 0 mg/L








































































































































































































































































































































































































































































































































































































































































































































































































































































































 day0123456789101112131415161718192021 sum
vessel No.                         
 Ytotal000000001000260038004300410 158
 Ydead0000000000000000000000 0
1Yalive000000001000260038004300410 158
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal00000000001600300042003700 125
 Ydead0000000000000000000000 0
2Yalive00000000001600300042003700 125
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000001500310040003800041 165
 Ydead0000000000000000000000 0
3Yalive000000001500310040003800041 165
 Saborted0000000000010000000000 1
 Palive1111111111111111111111  
                          
 Ytotal00000000080260034000390044 151
 Ydead0000000000000000000000 0
4Yalive00000000080260034000390044 151
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal000000000130280034000440046 165
 Ydead0000000000000000000000 0
5Yalive000000000130280034000440046 165
 Saborted0000000000000000001000 1
 Palive1111111111111111111111  













































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































 Ytotal00000000001700270035004200 121
 Ydead0000000000000000000000 0
6Yalive00000000001700270035004200 121
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal00000000001700330043004600 139
 Ydead0000000000000000000000 0
7Yalive00000000001700330043004600 139
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal00000000001600320040100370 126
 Ydead0000000000000000000000 0
8Yalive00000000001600320040100370 126
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          
 Ytotal00000000080027002300380038 134
 Ydead0000000000000000000000 0
9Yalive00000000080027002300380038 134
 Saborted0000000000000000001000 1
 Palive1111111111111111111111  
                          
 Ytotal00000000000110031003800330 113
 Ydead0000000000000000000000 0
10Yalive00000000000110031003800330 113
 Saborted0000000000000000000000 0
 Palive1111111111111111111111  
                          







Summary

Ytotal000000002529661222712217723160120121125111169 1397
 Ydead0000000000000000000000 0
 Yalive000000002529661222712217723160120121125111169 1397
 Saborted0000000000010000002000 3
 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 0-20000.0--
Day 9-1200
Day 19-2100
1.56Day 0-21.631.551.519897
Day 9-121.401.46
Day 19-211.561.52
3.13Day 0-23.263.123.0610098
Day 9-122.873.09
Day 19-213.083.05
6.3Day 0-26.486.126.0810097
Day 9-125.665.86
Day 19-216.096.58
12.5Day 0-212.812.412.310199
Day 9-1211.612.9
Day 19-2112.312.2
25Day 0-22524249997
Day 9-122424
Day 19-212424
a: based on 3 renewal period days.     
b: % of Mean Initial      
c: % of Nominal concentration      

 


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)201211221231241251261271281
11.56202212222232242252262272282
23.13203213223233243253263273283
36.25204214224234244254264274284
412.5205215225235245255265275285
525206216226236246256266276286
 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)
201 (2)a0<0.5<0.5-
201 (2)b0<0.5
202 (2)a1.561.651.63104
202 (2)b1.561.63
203 (2)a3.133.273.26104
203 (2)b3.133.26
204 (2)a6.256.546.48104
204 (2)b6.256.43
205 (2)a12.512.712.8102
205 (2)b12.512.8
206 (2)a252525100
206 (2)b2525

(1) Calculation is based on non-rounded values
(2) Sample production: Sep 22, 2021; Sampling on: Sep 22 2021. Samples were frozen on sampling date and analyzed on Sep 23, 2021.


 


 


 



























































































































































































































Summarized results of the second set of samples  
      
Sample namePreparationExpected contentFound content Mean contentMean recovery
  [mg/L][mg/L][mg/L] (1)[%] (1)
211 (2)a0<0.5<0.5-
211 (2)b0<0.5
212 (2)a1.561.551.55100
212 (2)b1.561.56
213 (2)a3.133.123.12100
213 (2)b3.133.13
214 (2)a6.256.036.1298
214 (2)b6.256.21
215 (2)a12.512.412.499
215 (2)b12.512.4
216 (2)a25242496
216 (2)b2524
221 (2)a0<0.5<0.5-
221 (2)b0<0.5
222 (2)a1.561.681.65106
222 (2)b1.561.61
223 (2)a3.133.093.13100
223 (2)b3.133.17
24 (2)a6.256.286.26100
24 (2)b6.256.24
25 (2)a12.512.212.398
25 (2)b12.512.5
26 (2)a25252599
26 (2)b2525
      
(1) Calculation is based on non-rounded values  
(2) Sample production: Sep 22, 2021; Sampling on: Sep 24 2021. Samples were frozen on sampling date and analyzed on Sep 29, 2021. 

 


 


 















































































































































Summarized results of the third set of samples  
      
Sampling dayPreparationExpected contentFound content Mean contentMean recovery
  [mg/L][mg/L][mg/L] (1)[%](1)
231 (2)a0<0.5<0.5-
231 (2)b0<0.5
232 (2)a1.561.391.4090
232 (2)b1.561.41
233 (2)a3.132.852.8792
233 (2)b3.132.89
234 (2)a6.255.755.6690
234 (2)b6.255.56
235 (2)a12.511.611.693
235 (2)b12.511.6
236 (2)a25232494
236 (2)b2524
      
(1) Calculation is based on non-rounded values  
(2) Sample production: Oct 01, 2021; Sampling on: Oct 01, 2021 
      

 


 


 



























































































































































































































Summarized results of the fourth set of samples  
      
Sample namePreparationExpected contentFound content Mean contentMean recovery
  [mg/L][mg/L][mg/L] (1)[%] (1)
241 (2)a0<0.5<0.5-
241 (2)b0<0.5
242 (2)a1.561.471.4693
242 (2)b1.561.44
243 (2)a3.133.103.0999
243 (2)b3.133.09
244 (2)a6.255.915.8694
244 (2)b6.255.81
245 (2)a12.513.112.9104
245 (2)b12.512.8
246 (2)a25242497
246 (2)b2524
251 (2)a0<0.5<0.5-
251 (2)b0<0.5
252 (2)a1.561.351.3587
252 (2)b1.561.35
253 (2)a3.132.752.8089
253 (2)b3.132.84
254 (2)a6.255.895.7893
244 (2)b6.255.67
255 (2)a12.511.811.894
255 (2)b12.511.8
256 (2)a25232393
256 (2)b2523
      
(1) Calculation is based on non-rounded values  
(2) Sample production: Oct 01, 2021; Sampling on: Oct 04, 2021. Samples were frozen on sampling date and analyzed on Oct 12, 2021 

 


 


 







































































































































Summarized results of the fifth set of samples  
      
Sampling dayPreparationExpected contentFound content Mean contentMean recovery
  [mg/L][mg/L][mg/L] (1)[%](1)
261 (2)a0<0.5<0.5-
261 (2)b0<0.5
262 (2)a1.561.561.56100
262 (2)b1.561.56
263 (2)a3.133.113.0898
263 (2)b3.133.04
264 (2)a6.256.176.0997
264 (2)b6.256.02
265 (2)a12.512.512.398
265 (2)b12.512.2
266 (2)a25242496
266 (2)b2524
      
(1) Calculation is based on non-rounded values  
(2) Sample production: Oct 11, 2021; Sampling on: Oct 11, 2021 

 


 


 


 

































































































































































































































Summarized results of the sixth set of samples  
      
Sample namePreparationExpected contentFound content Mean contentMean recovery
  [mg/L][mg/L][mg/L] (1)[%] (1)
271 (2)a0<0.5<0.5-
271 (2)b0<0.5
272 (2)a1.561.521.5297
272 (2)b1.561.51
273 (2)a3.133.043.0598
273 (2)b3.133.07
274 (2)a6.256.666.58105
274 (2)c6.256.59
274 (2)b6.256.50
275 (2)a12.512.212.298
275 (2)b12.512.3
276 (2)a25252497
276 (2)b2524
281 (2)a0<0.5<0.5-
281 (2)b0<0.5
282 (2)a1.561.531.5398
282 (2)b1.561.52
283 (2)a3.133.053.0598
283 (2)b3.133.06
284 (2)a6.256.086.0296
284 (2)b6.255.95
285 (2)a12.512.212.298
285 (2)b12.512.3
286 (2)a25252599
286 (2)b2525
      
(1) Calculation is based on non-rounded values  
(2) Sample production: Oct 11, 2021; Sampling on: JOct 13, 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:

  1. A Daphnia magna reproduction test (semi-static, 21 d) of the test item EDTA-ZnK2 (batch number: FC-C 11530) was conducted according to OECD 211 (2012) from 2021-Jul-07 to 2022-Mar-03, with the definitive exposure phase from 2021-Sep-22 to 2021-Oct-13 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-ZnK2 is a white solid. The aqueous media were prepared dissolving 50 mg of test substance in 2-L of test medium and stirring for approximately 10 min at ambient temperature. The lower test concentrations were prepared by dilution of this stock solution. Before the dilution was performed, the stock solution was checked for complete dissolution of the test substance. 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-ZnK2 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 purity of the test substance used in the test was 90.4%


LOEC = > 25 mg test substance/L or 22.6 mg a.i./L
NOEC = ≥ 25 mg test substance/L or 22.6 mg a.i./L

Description of key information

A Daphnia magna reproduction test (semi-static, 21 d) with the test item EDTA-ZnK2 was conducted according to OECD 211 under GLP conditions. This test result is result is read-across to EDTA-ZnNa2. The justification of this read-across is included in chapter 13 of the IUCLID file.


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-ZnK2 is a white solid. The aqueous media were prepared dissolving 50 mg of test substance in 2-L of test medium and stirring for approximately 10 min at ambient temperature. The lower test concentrations were prepared by dilution of this stock solution. Before the dilution was performed, the stock solution was checked for complete dissolution of the test substance. 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-ZnK2 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 purity of the test substance used in the test was 90.4%


LOEC = > 25 mg test substance/L or 22.6 mg a.i. EDTA-ZnK2/L which equals 20.9 mg a.i. EDTA-ZnNa2
NOEC = ≥ 25 mg test substance/L or 22.6 mg a.i. EDTA-ZnK2/L which equals 20.9 mg a.i. EDTA-ZnNa2

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
NOEC
Effect concentration:
20.9 mg/L

Additional information

Three reliable 21 d chronic toxicity studies for daphnids are available. A study from 2022 with EDTA-CuK2 which was performed in parallel to the study with the test substance (EDTA-ZnK2) and gave similar to the study with EDTA-CuK2 a 21d NOEC of 25 mg test substance/L.


There are also two older studies which both gave results in the same order of magnitude. The Key value used in the EU RAR 2004 is a study from BASF performed with EDTA-Na2H2 (CAS no 139-33-3). This study resulted similar as for EDTA-CuK2 and EDTA-ZnK2 in a 21d NOEC of 25 mg/L Recalculation towards EDTA-ZnK2 via MW ratio results in 431.8/336.21 x 25 = 32.1 mg/L