Aluminate(2),[[N,N'-1,2-ethanediylbis[N-(carboxymethyl)glycinato]](4-)-N,N',O,O',ON]hydroxy-,disodium

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

yes

Remarks:

No major (the temperature was not constant within ± 1°C for test concentrations 20.7, 45.5 and 100 mg/L. The test is still valid, as it was shown that validity criteria are met)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Test material form:

solid: flakes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:PREVOR, France - Batch number 070601
- Expiration date of the lot/batch:06 jun 2019
- Purity test date: 31 July 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:ambient (5 °C - 30 °C), dark, dry
- Stability under test conditions:stable
- Solubility and stability of the test substance in the solvent/vehicle: fully miscible


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:preparation of a gradient of solutions from a stock solution
- Final dilution of a dissolved solid:100, 45.5, 20.7, 9.39, 4.27 mg/L
- Final preparation of a solid:dilution into ISO water

FORM AS APPLIED IN THE TEST (if different from that of starting material): liquid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control (0 mg/l), 100, 45.5, 20.7, 9.39, 4.27 mg/L
- Sampling method: 21 ml sampled ( (aliquots of 20 ml + 1 ml nitric acid for stabilization).
- Sample storage conditions before analysis: stored between 1-10°C until analysis

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

- Method:Dilution into test medium (ISO test water) from 100 mg/L solution to 4.27 mg/l solution using the preceding solution (more concentrated). Prior using a solution as the dilution solution of an other, the mixture is homogenised by shaking. Afterwards the solution is clear and transparent.

- Controls: Test medium (ISO test water)

- Evidence of undissolved material (e.g. precipitate, surface film, etc.):None

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia Magna Straus
- Clone: V
- Source: Federal Environment Agency in Berlin/Germany
- Feeding during test: none
- Exposure to light:16 hours photoperiod /8 hours darkness daily

ACCLIMATION
- Acclimation conditions (same as test or not):same as test (preconditioned in ISO test water)
- Health during acclimation (any mortality observed):only offsprings (< 24 hours) are used for the test

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Daphnia magna was bred as single culture (1 daphnid per 100 mL) in Elendt M4 medium. The pH-value of the aerated water was within a range of 6.0 – 9.0. The dissolved oxygen was above 60 % saturation and the total hardness 140 - 250 mg/L (as CaCO3), corresponding to 7.8 - 14°dH. The animals were fed with single cell green algae (Desmodesmus subspicatus, formerly Scenedesmus subspicatus) at least three times a week.
The daphnids were reared at a temperature of 20 ± 2 °C in a climatic chamber with 16 hours of illumination and 8 hours of darkness. The medium was changed three times per week. A pipette was used to separate the young daphnids from the adults.
Freshly hatched daphnids less than 24 hours old were used for the test.

Study design

Test type:

static

Water media type:

other: ISO test water

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

13°dH (232 mg/L as CaCO3) (at test started, into the control, untreated test medium)

Test temperature:

19.4 – 21.7 °C

pH:

7.29 – 7.99

Dissolved oxygen:

≥ 8.6 mg/L

Salinity:

no

Nominal and measured concentrations:

yes

Details on test conditions:

TEST SYSTEM
- Test vessel: four 100 mL glass beakers per treatment, each filled with ~ 50 mL, one additional replicate for physico - chemical measurements without test organisms
- Type (delete if not applicable): closed with a glass plate
- Material, size, headspace, fill volume: 50 ml of test solution
- Aeration: none
- Type of flow-through (e.g. peristaltic or proportional diluter): none
- Renewal rate of test solution (frequency/flow rate):none
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 5 (4+1 for physico-chemical test)
- loading: ~ 10 mL of test solution for each animal

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:prepared on site facility - ISO test water
Ultra pure water with a conductivity < 10 µS·cm-1 was used. For 1L:
With: CaCl2.2H2O: 11.76g + MgSO4.7H2O: 4.93g + NaHCO3 2.59g + KCl 0.23g

- Culture medium different from test medium:Yes. Culture medium = Elendt M4 medium
- Intervals of water quality measurement: mesasured at test tart

OTHER TEST CONDITIONS
- Photoperiod:16 hours photoperiod /8 hours darkness daily


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : count of immobilised daphnids after 24 and 48 hours. All daphnids not able to swim within 15 seconds after gentle agitation of the test vessel were considered to be immobilised. If present, behavioural changes of daphnids were recorded at 24 and 48 hours after starting the test.


RANGE-FINDING STUDY
- Test concentrations: 0.1, 1.0 and 10.0 mg/l
- Results used to determine the conditions for the definitive study: No daphnia immobilized after 24 hours for any concentrations. 10% of daphnids immobilised after 48 hours at 10.0 mg/l

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

not determinable

Remarks:

Due to an inhibition below 50% the database was weak for probit analysis which hence was not performed

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

19.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Key result

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

9.39 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

9.39 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 5

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 78 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

mobility

Remarks on result:

not determinable

Remarks:

Due to an inhibition below 50% the database was weak for probit analysis which hence was not performed

Effect concentrations 6

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

14.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

mobility

Effect concentrations 7

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

8.64 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

mobility

Effect concentrations 8

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

8.64 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: After 24 hours the mobile daphnids at 45.5 and 100 mg/L were slightly sticky. After 48 hours some of the mobile daphnids at 9.39 mg/L were weak.

- Mortality of control: none

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: potassium dichromate (1.00 mg/L, 2.00 mg/L) under similar conditions and around same time period.
For 1.00 mg/l : 20% of immobilised daphnids after 24hours of exposure.
70% of immobilised daphnids after 48hours of exposure.
For 2.00 mg/l: 100% f immobilised daphnids after 24 and 48 hours of exposure.
The results indicate an EC50 (24 h) of the reference item potassium dichromate between 1.00 and 2.00 mg/L. Since the results are in accordance with the requirements of the OECD guideline 202 and fall within the historical data generated with the reference item at the testing facility, the daphnids were suitable for the determination of the toxicological effects of the test item.

Reported statistics and error estimates:

The value for EC50 (48 h) was determined by Probit analysis using linear max. likelihood regression. The evaluation of data was performed by ToxRat Professional 3.2.1.
The EC50 (24 h) was not determined statistically.
The NOEC was established based on the highest concentration at which the immobilisation is not higher than the allowed control immobilisation (≤ 10 % immobilisation).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

X300 was tested according to OECD 202 (2004) guideline for Toxicity to the Water Flea Daphnia magna Straus under Laboratory Conditions (Acute Immobilisation Test – Static) without any major deviation. According to the results of the test, the EC50 (48 h) was determined to be 19.3 mg/L (nominal) and 14.6 mg/L (measured). The corresponding NOEC (48 h) was 9.39 mg/L (nominal) and 8.64 mg/L (measured).

Executive summary:

Guidelines:       OECD 202 (2004).

Deviations to       

Guidelines:       No major (the temperature was not constant within ± 1°C for test concentrations 20.7, 45.5 and 100 mg/L. The test is still valid, as it was shown that validity criteria are met).

GLP:       Yes (certified laboratory)

Study Objective:       The objectives of this study were to determine the immobilisation effect of the test item X300 on the water flea Daphnia magna under worst-case exposure conditions, the no observed effect concentration (NOEC) and the effect median concentration (EC50), where possible.

Material and methods:

Test item:       X300, batch number: 070601, purity analysed: 92.70 % w/w.

Test organisms:       Daphnia magna Straus, Clone V, max. 24 hours old.

Test design:       Static dose-response test with twenty test organisms per treatment (4 replicates of 5 test organisms each) were used. The duration of the test was 48 hours.

Endpoints:       Endpoints reported are the EC50 and the NOEC after 24 and 48 hours.

Test rates:       A static main test with nominal concentrations of 100, 45.5, 20.7, 9.39, 4.27 mg/L, and control was performed.

Test conditions:       Temperature, pH-value and oxygen concentration of the test solutions measured after 0, 24 hours and 48 hours are reported. Hardness of the test medium (untreated control) was measured on the day of application.

Samples analysed:       Analytical samples taken at 0 hours (initial value) and 48 hours from aged test solutions were analysed from control and all test item concentrations.

Statistics:       The value for EC50 (48 h) was determined by Probit analysis using linear max. likelihood regression. The EC50 (24 h) was not determined statistically. The NOEC was established based on the highest concentration at which the immobilisation is not higher than the allowed control immobilisation (≤ 10 % immobilisation).

Dates of work:       14 Mar 2018 – 02 May 2018

 

Findings:       

Validity criteria:       Control immobilisation: The percentage of immobilisation should be ≤ 10 %. In this study the control immobilisation was 0 %.

Oxygen concentration: The dissolved oxygen concentration at the end of the test should be ≥ 3 mg/L in all test units. In this test, the dissolved oxygen concentration at the end of the test was ≥ 8.6 mg/L.

Test conditions:       The total hardness (as CaCO3) of the test medium (untreated control) was determined to be 13°dH (232 mg/L CaCO3); the mean pH-value of the untreated control was determined to be 7.59 ± 0.26 (Std. Dev.), the mean temperature of the control and all test item concentrations was measured to be 20.5 ± 0.9 °C (Std. Dev.) and the mean oxygen concentration was determined to be 8.9 ± 0.1 mg/L (Std. Dev.).

Analytical Results:       The initial measured content of active ingredient was between 82 and 84 % of nominal and the aged measured content was between 60 and 103 % of nominal. Therefore toxicological endpoints were evaluated using the nominal and the actual concentrations (based on the geometric mean of each measured concentration) of the test item.

Statistical Results:       EC50 and NOEC-values of daphnids exposed to the test item evaluated using nominal and actual concentrations

EC50 - X300 [mg/L] (nominal - 48h)    19.32 mg/l   

EC50 - X300 [mg/L] (geometric mean from measured - 48h): 14.62 mg/l

NOEC - X300 [mg/L] (nominal - 48h)    9.39 mg/l   

NOEC - X300 [mg/L] (geometric mean from measured - 48h): 8.64 mg/l

Conclusions:       According to the results of the test, the EC50 (48 h) was determined to be 19.3 mg/L (nominal) and 14.6 mg/L (measured). The corresponding NOEC (48 h) was 9.39 mg/L (nominal) and 8.64 mg/L (measured

).