Potassium hydrogen tartrate

Toxicity to soil macroorganisms except arthropods

Administrative data

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Remarks:

Earthworm Reproduction Test on Eisenia fetida on L(+) sodium potassium tartrate tetrahydrate

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Start of experimentation date: 26/09/2022
End of experimentation date: 25/01/2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2023

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

Reference substance 001

Cas Number:

6381-59-5

Test material form:

solid: particulate/powder

Remarks:

white

Details on test material:

Batch number 12001378620

Specific details on test material used for the study:

Internal number reference: Element_essai_22_0111.
Test item was stored at a mean temperature of 22°C, in a dry place.

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

Based on the results of the no-GLP range-finding test (RRCo-001309_01), the definitive test was performed at the following nominal concentrations of test item: control, 119.1, 238.1, 476.3, 952.5, 1905 mg.kg-1 (dry mass of soil), corresponding to 0, 62.5, 125, 250, 500, 1000 mg.kg-1 (dry mass of soil) of tartrate anion.
The tests soils were prepared using the pre-humidified artificial soil by direct addition of test/reference item stock solution into the volume of deionised water necessary to reach 60% of the WHC.
Eight replicates for control condition and four replicates for test item conditions were performed.
Reference item control, using boric acid, was carried out at the nominal concentrations of 400 and 600 mg.kg-1 (dry mass of soil) in the same time as the test. Two replicates for each concentration were performed.

Test substrate

Vehicle:

yes

Remarks:

Test item solutions were prepared in ultra pure water.

Details on preparation and application of test substrate:

Test item stock solution (SS) preparation:
- Name of SS: SM1_ecotox_22_0413
- Mass of test item (g): 11.4145
- Volume ultra pure water (mL): 1000
- Final concentration (mg.mL-1) 11.4145

Test organisms

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

The organisms used for this study were adult earthworms with clitellum of the species Eisenia fetida. The worms were selected from a synchronised culture with a relatively homogeneous age structure. Earthworms used in the study were selected without bias and earthworms exhibited poor health or damaged were rejected. They were acclimated one day before the exposure with the type of artificial soil and the test conditions used for the test. The mean weight and the 95% confidence interval on a random sample of 20 worms were calculated to determine the homogeneity of the population: mean individual weight was 474.4 mg (95% CI 444.2-504.7 mg). The test population was considered homogeneous because the average weight of the worms per replicate was within the calculated range.

Study design

Study type:

laboratory study

Substrate type:

artificial soil

Remarks:

Sieved sphagnum peat (10%) was mixed thoroughly with quartz sand (69.7%) and kaolin clay (20%). pH was adjusted by addition of CaCO3 (0.3%). The day before starting exposure, the soil was pre-humidified with deionised water to reach 30% of the WHC.

Limit test:

no

Total exposure duration:

56 d

Post exposure observation period:

20 minutes after the second 28-day period.

Test conditions

Test temperature:

20 ± 2°C

pH:

5.5 - 5.7

Moisture:

The water content of the soil substrate (between 40 % and 60% of the maximum water holding capacity) in the test containers is maintained throughout the test by re-weighing the test containers periodically.

Details on test conditions:

Each test vessel was filled with artificial soil (ca. 500 g dry mass of soil) moistened to 60% of its maximum water holding capacity with deionised water only for control or containing test item (at different concentrations) or containing reference item (at different concentrations).
The test or reference item was uniformly incorporated in the artificial soil and then 10 earthworms, previously acclimated for 24 hours in artificial soil identical to the one used in the test and rapidly rinsed with deionised water, were placed randomly in each test vessel.
To ensure easy migration of earthworms, the artificial soil was not compressed in the test vessels.
The test vessels were transparent and closed with a perforated lid to permit gaseous exchange between substrate and the atmosphere and access to light whilst preventing the worms for escaping and to prevent the test substrate from drying. They were placed under the test conditions for 56 days.
The worms were fed a first time one day after their introduction into the test vessels. About 5 g of food (pasteurized rabbit manure) was spread on the soil surface of each test vessel and moistened with deionised water (5 mL per test vessel). Subsequently, food was provided once a week for the first 28 days.
The water content was determined at the beginning and at the end of the test in each treatment group. Once a week, each container has been weighed to ensure that the water content of the soil substrate remains between 40% and 60% of the maximum water holding capacity and losses were compensated by deionised water.
The test was performed in a room at 20 ± 2°C under a controlled light-cycle of 16 hours of light and 8 hours of dark, with a light intensity of 400 to 800 lux. Moisture content and pH measurements of the soil were performed at the start and the end of the test.
On one sample for the control and one sample of each concentration of the test item, the pH was measured at the beginning and at the end of the test in each treatment group.
Mortality was assessed 28 days after the introduction of the worms by emptying the test substrate from the containers and by extracting the worms from the test substrate. All adult worms found alive were removed from the test vessels, observed for behaviour and morphology, counted and weighed (the worms were washed with deionised water and blotted dry before being weighed). All worms not found at that time were recorded as dead. The percentage of earthworm survival in each replicate was calculated for each concentration. Body weight of live adults in each replicate after the first 28 days was compared with their initial weight.
After evaluation of the 28-day mortality, the test substrate (minus the adult earthworms) was reintegrated into the test vessels respectively. The soil was then incubated for an additional 28 days under the same test conditions with the exception of feeding since a unique feeding (approximately 5 g) was introduced at the initiation of this second test phase.
At the end of this second 28-day period, the number of juveniles hatched from the cocoons in the test substrate was determined by placing the test units in a water bath at a temperature of about 40°C to 60°C. After a period of about 30 minutes, the juvenile earthworms appearing on the soil surface were removed and counted. The soil substrate was then quickly checked for non-hatched cocoons.
The mean number of juveniles produced per replicate in the test and toxic reference item treatment was compared with results of the control data.
Endpoints and if applicable their respective confidence limits were obtained by calculation according to appropriated statistics test for each response variable. ECx, NOEC and LOEC were also determined.

Nominal and measured concentrations:

Control, 119.1 mg/kg, 238.1 mg/kg, 476.3 mg/kg, 952.5 mg/kg, 1905 mg/kg (dry mass of soil) of sodium potassium tartrate tetrahydrate, corresponding to 0 mg/kg, 62.5 mg/kg, 125 mg/kg, 250 mg/kg, 500 mg/kg, 1000 mg/kg (dry mass of soil) of tartrate anion.

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

> 1 905 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 2

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

1 905 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: body weight change

Effect concentrations 3

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

1 905 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 4

Duration:

56 d

Dose descriptor:

EC10

Effect conc.:

1 854.6 mg/kg soil dw

95% CI:

800.5 - 8 925.4

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: estimated

Effect concentrations 5

Duration:

56 d

Dose descriptor:

other: EC20

Effect conc.:

> 1 905 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: estimated

Effect concentrations 6

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

> 1 905 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: estimated

Effect concentrations 7

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: expressed as tartrate anion

Basis for effect:

mortality

Effect concentrations 8

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: expressed as tartrate anion

Basis for effect:

other: body weight change

Effect concentrations 9

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: expressed as tartrate anion

Basis for effect:

reproduction

Effect concentrations 10

Duration:

56 d

Dose descriptor:

EC10

Effect conc.:

973.5 mg/kg soil dw

95% CI:

420.2 - 4 685.5

Nominal / measured:

nominal

Conc. based on:

other: expressed as tartrate anion

Basis for effect:

reproduction

Remarks on result:

other: estimated

Effect concentrations 11

Duration:

56 d

Dose descriptor:

other: EC20

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: expressed as tartrate anion

Basis for effect:

reproduction

Remarks on result:

other: estimated

Effect concentrations 12

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: expressed as tartrate anion

Basis for effect:

reproduction

Remarks on result:

other: estimated

Results with reference substance (positive control):

Results with reference substance (boric acid at 400 and 600 mg/kg dry soil) on mortality, total mass of worms and number of juveniles produced are displayed in the tables below.

Reported statistics and error estimates:

A statistical analysis has been performed to estimate the NOEC values for body weight change and for reproduction. A Shapiro-Wilk test was used to assess data normality and Bartlett’s test for homogeneity of variance. To check if a significant difference exists between concentrations and control, a pairwise t-test (parametric test) was performed. In all cases p values were > 0.05.

Any other information on results incl. tables

Validity of the study

The study was valid since mean mortality of the adults in the control was ≤ 10 % (actual value = 10%) after the initial 28 days; the number of juveniles was ≥ 30 in each control replicate (minimum actual value = 118) at the end of the test and the control coefficient of variation of reproduction was ≤ 30% at the end of the test (actual value = 12.1 %).

 

Detailed mortality data

The mean mortality of Eisenia fetida worms at 28 days is presented in the table below.

ID	Treatment / Nominal concentrations	Replicates	Number of living adults at the beginning	Number of living adults after 28 days	Mortality (%)	Corrected mortality (%)
C01	Control (Eigth replicates)	R01	10	8	10.0	0.0
		R02	10	10
		R03	10	6
		R04	10	10
		R05	10	10
		R06	10	9
		R07	10	9
		R08	10	10
C02	119.1 mg test item.kg-1 (dry mass of soil)	R01	10	8	12.5	2.8
		R02	10	10
		R03	10	7
		R04	10	10
C03	238.1 mg test item.kg-1 (dry mass of soil)	R01	10	10	7.5	-2.8
		R02	10	9
		R03	10	10
		R04	10	8
C04	476.3 mg test item.kg-1 (dry mass of soil)	R01	10	10	2.5	-8.3
		R02	10	9
		R03	10	10
		R04	10	10
C05	952.5 mg test item.kg-1 (dry mass of soil)	R01	10	10	0.0	-11.1
		R02	10	10
		R03	10	10
		R04	10	10
C06	1905 mg test item.kg-1 (dry mass of soil)	R01	10	10	17.5	8.3
		R02	10	7
		R03	10	7
		R04	10	9
C07	400 mg Boric acid.kg-1 (dry mass of soil)	R01	10	9	5.0	-5.6
		R02	10	10
C08	600 mg Boric acid.kg-1 (dry mass of soil)	R01	10	7	20.0	11.1
		R02	10	9

 

Detailed data on total mass of worms

The total mass of adults worms at the beginning (10 individuals/replicate) and after 28 days (living individuals /replicate) in each test container are presented in the table below as well as the body weight change (W in%) of Eisenia fetida worms after 28 days. 

ID	Treatment / Nominal concentrations	Replicates	Total mass of adults (mg) at start	Mean weight of individual adults(mg) at start	Total mass of living adults (mg) after 28 days	Mean weight of individual living adults after 28 days	W (body weight change) per replicate in %	Mean W (body weight change) in %
C01	Control (Eigth replicates)	R01	4609	460.9	5410	676.3	46.74	44.72
		R02	4228	422.8	6483	648.3	53.35
		R03	4739	473.9	3990	665.0	40.32
		R04	4682	468.2	6463	646.3	38.05
		R05	4880	488.0	6283	628.3	28.77
		R06	4493	449.3	6471	719.0	60.02
		R07	4626	462.6	6132	681.3	47.30
		R08	4551	455.1	6517	651.7	43.20
C02	119.1 mg test item.kg-1 (dry mass of soil)	R01	4517	451.7	5096	637.0	41.04	29.08
		R02	4789	478.9	5473	547.3	14.28
		R03	4348	434.8	3734	533.4	22.66
		R04	4724	472.4	6535	653.5	38.33
C03	238.1 mg test item.kg-1 (dry mass of soil)	R01	5623	562.3	6423	642.3	14.23	37.00
		R02	5120	512.0	5908	656.4	28.20
		R03	4628	462.8	6823	682.3	47.43
		R04	4744	474.4	6001	750.2	58.14
C04	476.3 mg test item.kg-1 (dry mass of soil)	R01	4817	481.7	5511	551.1	14.41	22.62
		R02	3712	371.2	5004	556.0	49.80
		R03	5298	529.8	5804	580.4	9.56
		R04	4500	450.0	5253	525.3	16.71
C05	952.5 mg test item.kg-1 (dry mass of soil)	R01	4937	493.7	5665	566.5	14.73	17.49
		R02	6242	624.2	6569	656.9	5.24
		R03	3960	396.0	4985	498.5	25.91
		R04	5279	527.9	6549	654.9	24.06
C06	1905 mg test item.kg-1 (dry mass of soil)	R01	5650	565.0	7108	710.8	25.81	40.87
		R02	4451	445.1	4955	707.9	59.04
		R03	4776	477.6	4870	695.7	45.67
		R04	4587	458.7	5488	609.8	32.96
C07	400 mg Boric acid.kg-1 (dry mass of soil)	R01	5566	556.6	5458.5	606.5	8.97	6.86
		R02	5136	513.6	5379.4	537.9	4.74
C08	600 mg Boric acid.kg-1 (dry mass of soil)	R01	5372	537.2	3877.3	553.9	3.12	2.48
		R02	5452	545.2	4997.2	555.2	1.84

 

Detailed data on juveniles produced

The number of juveniles produced and the effect on fecundity compared to the control (%) are presented in the table below.

ID	Treatment	Replicates	Number of    juveniles produced in each test container	Mean Number of    juveniles produced per treatment	Coefficient of     variation (%)	% effect on the fecundity compared to the control
C01	Control	R01	104	118.0	12.1	NA
		R02	121
		R03	116
		R04	142
		R05	111
		R06	114
		R07	135
		R08	101
C02	119.1 mg test item.kg-1 (dry mass of soil)	R01	122	114.3	6.8	3.2
		R02	104
		R03	113
		R04	118
C03	238.1 mg test item.kg-1 (dry mass of soil)	R01	126	130.5	12.3	-10.6
		R02	111
		R03	149
		R04	136
C04	476.3 mg test item.kg-1 (dry mass of soil)	R01	125	126.3	8.9	-7.0
		R02	132
		R03	111
		R04	137
C05	952.5 mg test item.kg-1 (dry mass of soil)	R01	132	135.8	18.0	-15.0
		R02	124
		R03	116
		R04	171
C06	1905 mg test item.kg-1 (dry mass of soil)	R01	135	107.0	20.9	9.3
		R02	95
		R03	84
		R04	114
C07	400 mg Boric acid.kg-1 (dry mass of soil)	R01	71	67.00	8.44	43.2
		R02	63
C08	600 mg Boric acid.kg-1 (dry mass of soil)	R01	44	42.00	6.73	64.4
		R02	40

NA= Not applicable

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effects of the test item at the nominal concentrations of 0, 119.1, 238.1, 476.3, 952.5 and 1905 mg.kg-1 (dry mass of soil) - corresponding to 0, 62.5, 125, 250, 500, 1000 mg.kg-1 (dry mass of soil) of tartrate anion - to Eisenia fetida were investigated under laboratory conditions.
The endpoints resulting of this test after 28 and 56 days of exposition are the following:
- LC50 (28 days) > 1905 mg/kg dry soil (corresponding to 1000 mg/kg dry soil of tartrate anion)
- NOEC reproduction after 56 days = 1905 mg/kg dry soil (corresponding to 1000 mg/kg dry soil of tartrate anion).

Executive summary:

The aim of this study was to assess the effects of the test item L(+) sodium potassium tartrate tetrahydrate in artificial soil on the mortality and reproductive output (and other sub-lethal end points) of the earthworm species Eisenia fetida, according to OECD guideline 222 (2016), over a period of eight
weeks.
The effects of the test item at the nominal concentrations of 0, 119.1, 238.1, 476.3, 952.5 and 1905 mg.kg-1 (dry mass of soil) - corresponding to 0, 62.5, 125, 250, 500, 1000 mg.kg-1 (dry mass of soil) of tartrate anion - to Eisenia fetida were investigated under laboratory conditions.
The endpoints resulting of this test after 28 and 56 days of exposition are the following:
- LC50 (28 days) > 1905 mg/kg dry soil (corresponding to 1000 mg/kg dry soil of tartrate anion)
- NOEC reproduction after 56 days = 1905 mg/kg dry soil (corresponding to 1000 mg/kg dry soil of tartrate anion).