Dipotassium [[N,N'-ethylenebis[N-(carboxylatomethyl)glycinato]](4-)-N,N',O,O',ON,ON']zincate(2-)

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-10-11 to 2018-10-26 with the definitive exposure time from 2018-10-11 to 2018-10-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

The respective test item amount was weighed out for a stock solution (nominal: 4440 mg/200 mL, actual: 4440.3 mg/200 mL) and dispersed in demineralised water. The stock solution was further diluted to the respective application solutions. The application solution and additional demineralised water were added to the artificial soil to adjust the humidity of the artificial soil to a moisture of 54 % of the WHCmax. Subsequently, the test medium was thoroughly mixed to ensure a homogenous distribution and about 555 g SDW were filled into each test vessel.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Eisenia fetida (Annelida, Lumbricidae)
- Source: Breeding stock culture maintained at the test facility
- Age at test initiation (mean and range, SD): Adult
- Weight at test initiation (mean and range, SD): 0.47 +/- 0.04 g

ACCLIMATION
- Acclimation period: 2 days
- Acclimation conditions (same as test or not): same as test
- Health during acclimation (any mortality observed): no mortality observed

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

14 d

Test temperature:

18 - 22 °C

pH:

Test item concentration
[mg/kg SDW]
pH-value

day 0 day 14
Control 6.31 5.75
10 6.37 5.75
31.6 6.29 5.75
100 6.30 5.79
316 6.34 5.81
1000 6.34 5.78

Moisture:

Test item concentration
[mg/kg SDW]
Moisture [%] of DW
[g /100 g SDW]
day 0 day 14
Control 24.8 23.5
10 24.8 23.6
31.6 24.3 23.6
100 24.5 23.6
316 25.2 23.7
1000 25.1 24.0

Details on test conditions:

TEST SYSTEM
- Test containers Glass vessels (volume 1.5 L, used for preserving) were used. During the test, transparent and perforated lids enabled sufficient gas exchange and light input and prevented drying of the test substrate.
- Amount of soil or substrate: 555 g soil dry weight per replicate
- No. of organisms per container (treatment): 10 per replicate
- No. of replicates per treatment group: 4
- No. of replicates per control: 4

Artificial soil:
- Artificial soil consisted of the following components:
- 10 % Peat, air-dried and finely ground
- 20 % Kaolin, kaolinite content > 30 %
- 69 % Air-dried quartz sand (sand with > 50 % particles size of 0.05 to 0.2 mm)
- 0.40 % calcium carbonate (CaCO3) to adjust the pH to 6.0 ± 0.5
The soil moisture content and the WHCmax were determined prior to experimental starting.
Two days before earthworm insertion into the test vessels the artificial soil (17 kg) was pre-moistened with 1425 g deionised water to achieve a moisture of 27 % of the WHCmax of the artificial soil. The moistened soil was kept covered to prevent evaporative water losses prior to the start of exposure.

OTHER TEST CONDITIONS
- Photoperiod: 24 h
- Light intensity: 536 ± 77.9

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The live weight of the earthworms was determined individually on the day of application (day 0) and on day 14.
The mean weight change was assessed on day 14 after application.
Mortality, behaviour and morphological changes of the earthworms were recorded 7 and 14 days after application.

VEHICLE CONTROL PERFORMED: no

TEST CONCENTRATIONS
- Spacing factor for test concentrations: √10
- Justification for using less concentrations than requested by guideline:
- Range finding study
A preliminary range finding test was performed with 3 test item concentrations.
2 replicates without the addition of food with 10 earthworms each.

Range Finding Test (14 days): Mortality and Change of Live Weight
Concentration Mortality of adult earthworms Change of Live weight
[mg test item/kg SDW] [%] [%]
Control 0 -11
10 0 -10
100 0 -18
1000 50 -35

Nominal and measured concentrations:

Nominal: 10 – 31.6 – 100 – 316 – 1000 mg/kg soil dry weight

Reference substance (positive control):

yes

Remarks:

2-Chloroacetamide

Key result

Duration:

14 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Mortality, behaviour, pathological symptoms

Key result

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Mortality, behaviour, pathological symptoms

Key result

Duration:

14 d

Dose descriptor:

LOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Change of body weight

Key result

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

316 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Change of body weight

Key result

Duration:

14 d

Dose descriptor:

EC10

Effect conc.:

430 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Change of body weight

Remarks on result:

other: CI (95%) = 301 - 615 mg/kg soil DW

Key result

Duration:

14 d

Dose descriptor:

other:

Remarks:

EC20

Effect conc.:

634 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Change of body weight

Remarks on result:

other: CI (95%) = 441 - 921 mg/kg soil DW

Key result

Duration:

14 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Change of body weight

Details on results:

- Mortality at end of exposure period:
No statistically significant mortality was observed in all test item concentrations after 14 days of exposure. Consequently no LCx values were determined.
- Total mass of adults at beginning of test:
Control: 19.1 g
10 mg/kg soil DW: 19.0 g
31.6 mg/kg soil DW: 18.9 g
100 mg/kg soil DW: 19.0 g
316 mg/kg soil DW: 19.0 g
1000 mg/kg soil DW: 19.0 g
- Changes in body weight of live adults (% of initial weight) at end of exposure period:
In terms of body weight loss, the NOEC was determined to be 316 mg/kg soil dry weight and LOEC was determined to be 1000 mg/kg soil dry weight.
- Morphological abnormalities:
No pathological symptoms or changes in the earthworm behaviour were observed in the control as well as in the test item concentrations 10 to 316 mg/kg soil dry weight after 7 days of exposure. In the test item concentration 316 mg/kg soil dry weight after 14 days and
1000 mg/kg soil dry weight after 7 and 14 days significant pathological symptoms were observed.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels:
NOEC and LC-Value after 14 d for the Reference Item 2-Chloroacetamide
LC-Value /
NOEC Concentration
[mg reference item/kg SDW] 95 % - Confidence Interval
[mg reference item/kg SDW]
NOEC 30 –
LC50 42.3 n.d.*
*) not determined due to mathematical reasons (3 reference item concentrations)

Reported statistics and error estimates:

LC values No LCx values were determined since no statistically significant mortality occurred.

EC values The EC10/20/50 values were determined by Analysis of Variance and Test for Lack of Fit for the 3-param. normal CDF.

NOEC, LOEC and statistical analyses
The NOEC/LOEC for mortality was determined using a Multiple
Sequentially-rejective Fisher Test After Bonferroni-Holm for determination of statistically significant differences in mortality compared to the control. When running the test, a Qualitative Trend Analysis by Contrasts and a Tarone’s Test Procedure for signs of extra-binomial variance were done first.
Statistically significant differences in biomass compared to the control were determined using a Williams Multiple Sequential t-test Procedure. When running the test, a Shapiro-Wilk’s Test on Normal Distribution, a Levene’s Test on Variance Homogeneity (with Residuals) and Trend analysis by Contrasts (Monotonicity of Rate/Response) were done first. The alpha-value (acceptable probability of incorrectly concluding that there is a difference) is alpha = 0.05.

Earthworm Mortality in [%] after 7 and 14 Days of Exposure

								Mortality [%]
Test item concentration [mg/kg SDW]	Day 7									Day 14
	1		2		3		4		MV	1	2	3	4	MV
Control	0		0		0		0		0	0	0	0	0	0
10	0		0		0		0		0	0	0	0	0	0
31.6	0		0		0		0		0	0	0	0	0	0
100	0		0		0		0		0	0	0	0	0	0
316	0		0		0		0		0	0	0	0	0	0
1000	0		0		0		0		0	10	30	0	0	10

MV = mean value

Earthworm Behaviour and Pathological Symptoms after 7 and 14 Days of Exposure

Test item concentration [mg product/kg SDW]	Effect	Replicate
		1	2	3	4
		Day 7
Control	A)	10/10	10/10	10/10	10/10
3.16	A)	10/10	10/10	10/10	10/10
10	A)	10/10	10/10	10/10	10/10
31.6	A)	10/10	10/10	10/10	10/10
100	A)	10/10	10/10	10/10	10/10
316	A)	10/10	10/10	10/10	10/10
1000	A)	10/10	7/10	10/10	10/10
	D)	-	3/10	-	-
		Day 14
Control	A)	10/10	10/10	10/10	10/10
3.16	A)	10/10	10/10	10/10	10/10
10	A)	10/10	10/10	10/10	10/10
31.6	A)	10/10	10/10	10/10	10/10
100	A)	10/10	10/10	10/10	10/10
316	A) D)	10/10 -	10/10 -	9/10 1/10	8/10 2/10
1000	A) B,D,G)	7/10 -	4/10 -	7/10 1/10	6/10 -
	D)	2/10	3/10	2/10	4/10
	H)	1/10	3/10	-	-

A) no obvious pathological symptoms                                           

B) no reaction to touching

D) spontaneous segmentation and separation                              

G) ulcer and skin bleedings

H) dead earthworm

Mean Live Weight and Mean Loss of Body Weight

	Mean live weight
Test item  concentration  [mg product/kg SDW]	Test start MV±SD [g/worm]	Test end MV±SD [g/worm]	Inhibition [%]	Statistically significant vs.  Control
Control	0.48 ± 0.03	0.41 ± 0.04	14.6	–
10	0.47 ± 0.04	0.42 ± 0.04	10.6	No
31.6	0.47 ± 0.05	0.41 ± 0.04	12.8	No
100	0.47 ± 0.05	0.42 ± 0.05	10.6	No
316	0.47 ± 0.04	0.40 ± 0.07	14.9	No
1000	0.47 ± 0.04	0.29 ± 0.10	38.3	Yes

MV = Mean value           SD = Standard deviation

Validity criteria fulfilled:

yes

Conclusions:

No statistically significant mortality was observed in all test item concentrations after 14 days of exposure. Therefore, the NOEC was determined to be ≥ 1000 mg/kg soil dry weight and LOEC was determined to be > 1000 mg/kg soil dry weight.
In terms of body weight loss, the NOEC was determined to be 316 mg/kg soil dry weight and LOEC was determined to be 1000 mg/kg soil dry weight.

Executive summary:

The acute effects ofEDTA-ZnK2(Batch:FC-C11530)on the earthwormEisenia fetidawere determined according to the OECD Guideline 207 at the test facility in 31157 Sarstedt, Germany from 2018-10-11 to 2018-10-26 with the definitive exposure time from 2018-10-11 to 2018-10-25. The study was conducted under static conditions for 14 days with the test item concentrations 10 – 31.6 – 100 – 316 and 1000 mg/kg soil dry weight mixed into artificial soil containing 10 % peat. Artificial soil without test item was used as control. 40 test organisms with an individual weight between 0.38 - 0.54 g, divided into four replicates, were tested per test item concentration and control.

No statistically significant mortality was observed in all test item concentrations after 14 days of exposure.Therefore, theNOEC was determined to be ≥ 1000 mg/kg soil dry weight and LOEC was determined to be > 1000 mg/kg soil dry weight. No LC_xvalues were determined since nostatistically significantmortality was observed.

In terms of body weight loss, the NOEC was determined to be 316 mg/kg soil dry weight and LOEC was determined to be 1000 mg/kg soil dry weight.

All validity criteria of the test guideline were fulfilled.

 

 Summary of all Effects

Effects	EDTA-ZnK2 [mg/kg soil dry weight]
LOEC Mortality, behaviour, pathological symptoms	> 1000
NOEC Mortality, behaviour, pathological symptoms	≥1000
LOEC Change of body weight	1000
NOEC Change of body weight	316
EC10body weight(confidence interval) EC20body weight(confidence interval) EC50body weight(confidence interval)	430 (301 – 615) 634 (441 – 921) > 1000

Description of key information

One acute toxicity study with earthworms is available for EDTA-ZnK2 (Winkelmann, 2019). The EC50 for weight was determined to be >1000 mg test item/kg dw. The active ingredient content of the test item was 90.4 %. The EC50 for weight based on active was thus >904 mg a.i./kg dw.

In terms of body weight loss, the NOEC was determined to be 316 mg/kg soil dry weight and LOEC was determined to be 1000 mg/kg soil dry weight. Corrected for active ingredient this results in a NOEC and LOEC of 285.6 and 904 mg/kg dw.      

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:

285.6 mg/kg soil dw

Additional information

The acute effects of EDTA-ZnK2 on the earthworm Eisenia fetida were determined according to the OECD Guideline 207. The study was conducted under static conditions for 14 days with the test item concentrations 3.16 – 10 – 31.6 – 100 – 316 and 1000 mg/kg soil dry weight mixed into artificial soil containing 10 % peat. Artificial soil without test item was used as control. 40 test organisms with an individual weight between 0.38 - 0.54 g, divided into four replicates, were tested per test item concentration and control.

No statistically significant mortality was observed in all test item concentrations after 14 days of exposure. Therefore, the NOEC was determined to be ≥ 1000 mg test item/kg soil dry weight (≥ 904 mg a.i./kg dw) and LOEC was determined to be > 1000 mg test item/kg soil dry weight ( > 904 mg a.i./kg dw). No LCx values were determined since no statistically significant mortality was observed.

In terms of body weight loss, the NOEC was determined to be 316 mg test item/kg soil dry weight (286 mg a.i./kg dw)

and LOEC was determined to be 1000 mg test item/kg soil dry weight (904 mg a.i./kg dw)..

All validity criteria of the test guideline were fulfilled.