Tetrasodium N,N-bis(carboxylatomethyl)-L-glutamate

Toxicity to soil macroorganisms except arthropods

Administrative data

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2-7-2015 till 28-8-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP conditions with chemical analysis

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

No

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Tetrasodium N,N-bis(carboxylatomethyl)-L-glutamate

EC Number:

257-573-7

EC Name:

Tetrasodium N,N-bis(carboxylatomethyl)-L-glutamate

Cas Number:

51981-21-6

Molecular formula:

C9H9NO8Na4

IUPAC Name:

tetrasodium 2-[bis(carboxylatomethyl)amino]pentanedioate

Constituent 2

Reference substance name:

N,N-bis(carboxymethyl)-L-glutamic acid tetra sodium salt

IUPAC Name:

N,N-bis(carboxymethyl)-L-glutamic acid tetra sodium salt

Test material form:

other: solution in water

Details on test material:

Chemical name: GLDA-Na4 aq. sol
Chemical name: L-glutamic acid N,N-diacetic acid tetrasodium salt
CAS No.: 051981-21-6
Batch Number: FC-C 11836
Active ingredient content: 47.5%
Appearance: Light yellow, clear liquid (max. 250 APHA)
Water solubility: Entirely soluble

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The concentration of the test item (CAS No. 051981-21-6) was confirmed at test start (day 0) by analytical verification of all test item solutions and the control using LC-MS/MS. After 28 and 55 days, mixed soil samples out of the replicates were taken for analysis (overall amount 10 – 20 g per treatment) just before any biological observation.

Test substrate

Vehicle:

no

Details on preparation and application of test substrate:

The respective test item amount for each concentration was weighed out, dissolved in demineralised water and added to the artificial soil. Additional demineralised water was added to adjust the humidity of the artificial soil to a moisture of 40 – 60 % of the WHCmax. Subsequently, the test medium was mixed thoroughly to ensure a homogenous distribution, and a soil amount corresponding to about 600 g soil DW was filled into each test vessel.

Test organisms

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

Test organism: Eisenia fetida (Annelida, Lumbricidae)
Reason for the selection Eisenia fetida is suitable for this kind of study and is one of the recommended species according to the guideline.
Source Breeding stock culture maintained at the test facility
Breeding Organisms of the species Eisenia fetida are bred at the test facility in covered plastic vessels containing potting compost
Synchronisation of the Earthworms of homogeneous age and body weight were used in earthworm population the definitive test. Synchronisation of the population was achieved by placing adult earthworms into breeding boxes and removing the adults after 4 weeks. Offspring from the remaining cocoons reached the adult age after 2 months at the earliest.
Feeding:
During breeding, the earthworms were fed with a litter of dried stinging nettle leaves and porridge oats. A sufficient amount of the food was provided depending on the feeding rate and the density of the earthworm population in the vessels.

Study design

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Test conditions

Test temperature:

20 ± 2 °C

Moisture:

Throughout the test, the replicates were weighed at weekly intervals. Water was added to compensate for individual evaporation losses and to maintain the soil moisture, which should not deviate by more than 10 % of the initial value at the end of the test. Additional moistening as described in Table 3 was performed in case the soil appeared too dry.

Details on test conditions:

Photoperiod 16 h; Light intensity 400 - 800 lx
Test units containing about 600 g DW of soil were round plastic boxes with an inner diameter of 15.0 cm, corresponding to a bottom surface area of 177 cm2, and a height of 14 cm. Transparent and perforated lids enabled sufficient gas exchange and light input and prevented the test substrate from drying.
The earthworms were fed with air-dried cattle manure delivered by LEHR- UND FORSCHUNGSGUT RUTHE (Institute of the University of Veterinary Medicine Hannover, Foundation), Schäferberg 1, D-31157 Sarstedt, Germany, at weekly intervals during the first 4 weeks as presented in Table 3. At test start and on day 28, the food was mixed carefully into the soil, on the other days the food was placed onto the soil surface.

Nominal and measured concentrations:

nominal test item concentrations 62.5, 125, 250, 500 and 1000 mg test item/kg soil DW – corresponding to the
geometric mean measured concentrations 3.74, 9.39, 15.4, 38.0 and 248 mg test item /kg soil DW

Reference substance (positive control):

yes

Remarks:

CARBENDAZIM

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

248 mg/kg soil dw

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

47.5% active ingredient

Basis for effect:

reproduction

Remarks:

& Growth

Effect concentrations 2

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

117.8 mg/kg soil dw

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks:

Based on 100% active ingredient

Basis for effect:

reproduction

Remarks:

& Growth

Details on results:

After 28 days of exposure in soil, no statistically significant earthworm mortalities, pathological symptoms or changes in the behaviour of adult earthworms were observed in any treatment. The average body weight of adult earthworms increased during the first 28 days in all treatments without statistically significant differences.
After a further four weeks, the reproduction rate (mean number of juveniles produced per replicate) was 109 in the control and ranged from 92 to 162 in the test item treatments. Despite a distinct promotion of reproduction in the geometric mean measured test item concentration 15.4 mg/kg soil DW, no statistically significant reduction of reproduction compared to the control was observed in any treatment.
Consequently, the NOEC of the test item concerning growth and reproduction was determined to be 248 mg/kg soil DW. No ECx-values were calculated since no statistically significant mortality and reduction of body weight gain or reproduction occurred.

Results with reference substance (positive control):

According to OECD 222, significant effects of Carbendazim were observed between 1 and 5 mg a.s./kg soil DW.

Reported statistics and error estimates:

Adult Mortality
The arithmetic mean and NOEC were determined. No EC-values for mortality were calculated as no significant effects were observed.

Growth, Reproduction
Significant differences in growth and reproduction were determined (stimulation) in comparison with the control group using Dunnett’s Multiple t-test Procedure, Multiple Sequentially Rejective Fisher Test and Multiple Sequentially-rejective Median Test. When running the test, Shapiro-Wilk’s Normality Test and Levene’s Equal Variance Test were done first. The alpha-value (acceptable probability of incorrectly concluding that there is a difference) is alpha = 0.05.
No ECx-values were calculated since no test item-related reduction of body weight gain or reproduction exceeding the respective thresholds occurred at the tested application rates. 
Software
The data presented in the tables of the report were computer-generated and have been rounded for presentation. Thus, manual re-calculation of the data based on the results presented in this report may result in minor deviations from these figures. Calculations were carried out using software
- Excel, MICROSOFT
- Sigma Plot, SPSS INC.
- TOXRAT PROFESSIONAL, TOXRAT SOLUTIONS GMBH

Any other information on results incl. tables

The method was validated according toSANCO/3029/99 rev.4 (2000) prior to the start of exposure.

The measured concentrations of the stock solutions at test start werebetween 96 and 109 %ofthe nominal values. Therefore, nominal concentrations were taken into account for geometric mean calculation. On day 28, the extracted test item concentrations in the soil were between 13 and 29 % of the nominal values. At the end of the study, in the highest test item treatment a concentration of 5 % of the nominal value could be detected. In all other treatments no test item concentration above the SysQL was found, but the concentrations were above the LOD (limit of detection) and therefore used for geometric mean calculations.

As in the course of the study improvements on the analytical soil sample measurements were done, an extraction efficiency test with spiked soil was performed, revealing an extraction efficiency of 68 to 108 % .

 

Table9:          Measured Concentrations and Percentages of the nominal Concentrations of the Test Item

Sampling date		2015-07-02		2015-07-30		2015-08-26
Start of analysis		2015-07-02		2015-07-31		2015-08-27
Nominal concentration		Dissolvine GL-47-S (CAS No.051981-21-6)
[mg/kg DW]	[mg/L] (stock solution)	Meas. conc. [mg/L]	%	Meas. conc. [mg/kg]	%	Meas. conc. [mg/kg]		%	Geometric mean measured test item concentration [mg/kg soil DW]^2)
1000	11906	11432	96	294	29	51.6^3)		5	248
500	5988	5843	98	108	22	1.02^1)	0		38.0
250	2995	2861	96	33.1	13	0.442^1)	0		15.4
125	1500	1633	109	18.9	15	0.351^1)	0		9.39
62.5	750	791	105	8.50	14	0.0987^1)	0		3.74
Control	-	< SysQL		< SysQL		< SysQL			-

 Measured concentration     = Measured concentration of the test item, dilution factor taken into account

%                          = Percentage of nominal concentration of the test item

SysQL                     =System Quantification Limit(0.5 mg/L test item)

1)                          = Values < SysQL but > LOD were calculated and taken into account for Geometric mean Calculation

2)                          = For Geometric mean calculation, nominal values at the start of the exposure (verified via stock solution analysis) were taken into account

3)                          = Reanalysed on 2015 -08 -28

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

All validity criteria specified by the test guidelines were fulfilled.

Conclusions:

The test is considered valid as all validity criteria have been fulfilled; Based on the test setup (GLP, Guideline study) and the extensive effort which has been paid to quantify the exposure not only at the start but also at the end of the study the results are considered to be reliable.

Executive summary:

The effects of the test item (CAS No. 051981-21-6) (Batch no.FC-C11836) on mortality, growth and the reproductive potential of the earthworm species Eisenia fetida (Annelida, Lumbricidae) were determined according to OECD Guideline 222 (2004) fromJuly 2^nd to August 28^th , 2015with the definitive exposure phase fromJuly 2^nd to August 26^th , 2015 at Dr.U.Noack-Laboratorien in D-31157 Sarstedt, Germany. The study was conducted over 8 weeks with the nominal test item concentrations 62.5, 125, 250, 500 and 1000 mg test item/kg soil DW – corresponding to the geometric mean measured test item concentrations 3.74, 9.39, 15.4, 38.0 and 248 mg test item /kg soil DW -, which were mixed into artificial soil containing 10 % peat. As a control, untreated artificial soil was used.Sexually mature earthworms were inserted into 8 control replicates and into 4 replicates per test item concentration (10 earthworms per replicate).They had individual body weights between 0.32 and 0.55 g at the start of exposure.

 

After 28 days of exposure in soil, no statistically significant earthworm mortalities, pathological symptoms or changes in the behaviour of adult earthworms were observed in any treatment. The average body weight of adult earthworms increased during the first 28 days in all treatments without statistically significant differences.

After a further four weeks, the reproduction rate (mean number of juveniles produced per replicate) was 109 in the control and ranged from 92 to 162 in the test item treatments. Despite a distinct promotion of reproduction in the geometric mean measured test item concentration 15.4 mg/kg soil DW, no statistically significant reduction of reproduction compared to the control was observed in any treatment.

Consequently, the NOEC of the test item concerning growth and reproduction was determined to be 248 mg/kg soil DW.

No EC_x-values were calculated since no statistically significant mortality and reduction of body weight gain or reproduction occurred.