Amides, adipic acid

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 10 March, 2020 to 25 March, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples, properly diluted with deionised water with low TOC contents, have been analysed by means of an integrated sampling system directly from vials containing 40 ml volume. For each sample under test the corresponding blank has been analysed.

Vehicle:

yes

Remarks:

culture medium

Test organisms (species):

Daphnia magna

Details on test organisms:

Characterization Test organism:
Age: Daphnia magna straus, Crustacean, Cladocera, 6-24 hours
Origin: Breeder in Eurofins Biolab Test Facility
N. of animals: 20 for each treated - 20 for control
N. of animals/beaker: 5
N. of replications: 4 for control and 4 for each treated concentration
N. of concentrations: 5 for range finding test and 1 for limit test

Maintenance Culture vessels: Glass Beaker (500 mL).
Maintenance in culture: Daphnie have been kept in reconstituted water in the following conditions:
Temperature : 20°C ± 2°C
Lightning: Photoperiod: 16 hours of light, 8 hours of darkness
Water hardness: 140- 250 mg CaCO3/L
Type of feeding: Green alga Pseudokirchneriel/ a subcapitata and Saccharomices cerevisiae
Water change: 3 times per week
Justification of assay system: Daphnia magna straus has been used for this test because is one of the recommended specie in OECD 202.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

140- 250 mg CaCO3/L

Test temperature:

20°C ± 2°C

pH:

7.83 - 7.98

Dissolved oxygen:

5.61 - 6.38 mg/L

Nominal and measured concentrations:

Range finding test: 0.1, 1, 10, 100 and 1000 mg/L (nominal)
Definitve test: 100 mg/L (nominal)

Details on test conditions:

Assay conditions
Assay duration: 48 ± 2 hours
Lightning: None during the course of the test.
Feeding: None during the course of the test.
Temperature : 20°C ± 2°C
Water: Reconstituted dilution water
Dissolved oxygen: > 3 mg/L
Hardness : between 140 and 250 mg CaCO3/L

Treatment
4 replicates with tested concentrations have been prepared, afterwards Daphnie have been added to the vessels of the assay sample. The control group have been treated in the same conditions without adding the test substance. Daphnie have been kept without light, aeration and feeding for the whole length of the assay. Animals have been kept under observation for a period of 48 hours.

Observations
After 24 and 48 hours from the beginning of the test, the immobilisation of the Daphnie have been evaluated verifying if the organisms, after light stirring of the vessels, were not capable to move in the following 15 seconds. Any abnormal behaviour or appearance, if present, have been recorded. pH, dissolved oxygen have been measured at the beginning and at the end of the test (after 48 hours) in the highest concentration in the treated group and in control. Temperature of the assay environment has been measured at the beginning and at the end of the test (after 48 hours).

Stability of the test substance
The results of organic carbon present in the test substance, at initial (t=0) and final timing test (48 h), corrected from the organic carbon present in the control have been used to verify the stability. The sample is considered stable if the test substance at the end of the test has been maintained within ±20% to the concentrations of organic carbon at the beginning of the test, according to OECD 202.

Validity criteria
The immobilisation of control animals must not be higher than 10% at the end of the test. The concentration of dissolved oxygen in the vessels ought to be higher than 3 mg/L during the assay . However, in any case, the concentration of dissolved oxygen must not be lower than 2 mg/L. pH must not change more than 1.5 units.

Equipments
- Multiparametric (pH-meter oxygen meter)
-Hood
-Dupla Test (KH) to measure total hardness
-Luxmeter
-Analytical scale
-Incubator
-Laboratory glassware
-MilliQ-Biocel A10
-Sterile graduated test tubes and pipettes
-Micropipettes
-Chronometer
-Magnetic stirrer TOC analyser
-Refrigerator

Reagents
Bihydrated calcium chloride, Eptahydrated magnesium sulphate, Acid sodium bicarbonate, Potassium chloride, Water MilliQ

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Validity criteria have been satisfied.
% Of immobilized Daphnie in control: 0

Daphnia immobilisation
The number of daphnia immobilized in the control and in the treated groups, in range finding and definitive test, has been reported. All daphnids survived to the end of the test. No abnormal behaviour or appearance has been observed.

Oxygen concentration
The results, for range finding and definitive test satisfied validity criteria.

pH and temperature
The results, for range finding and definitive test satisfied validity criteria.

Analytical verification of the concentration ofthe test substance
The test substance should be considered stable, because the TOC values, at the end of the test remain within the required range (80-120%) in comparison with the beginning measured values.

Results

Table N. 1: Number of immobilised Daphnie at 24 and 48 hours in treated (test substance) and control groups in the range finding test and in the definitive test

 

Range finding test

Control	24 hours	48 hours
Control replication N.1	0/5	0/5
Control replication N.2	0/5	0/5
Control replication N.3	0/5	0/5
Control replication N.4	0/5	0/5
Concentration 1:1000.00mgll
	24hours	48 hours
Treated Dilution 1-replication N.1	0/5	1/5
Treated Dilution 1-replication N.2	0/5	1/5
Treated Dilution 1-replication N.3	0/5	1/5
Treated Dilution 1-replication N.4	0/5	0/5
Concentration2: 100.00 mgll
	24 hours	48hours
Treated Dilution 2-replication N.1	0/5	0/5
Treated Dilution 2-replication N.2	0/5	0/5
Treated Dilution 2-replication N.3	0/5	0/5
Treated Dilution 2-replication N.4	0/5	0/5
Concentration 3 : 10.00 mg/L
	24 hours	48 hours
Treated Dilution 3-replication N.1	0/5	0/5
Treated Dilution 3-replication N.2	0/5	0/5
Treated Dilution 3-replication N.3	0/5	0/5
Treated Dilution 3-replication N.4	0/5	0/5
Concentration 4: 1.00 mg/L
	24 hours	48 hours
Treated Dilution 4-replication N.1	0/5	0/5
Treated Dilution 4-replication N.2	0/5	0/5
Treated Dilution 4-replication N.3	0/5	0/5
Treated Dilution 4-replication N.4	0/5	0/5
Concentration 5:0.10 mg/L
	24 hours	48 hours
Treated Dilution 5-replication N.1	0/5	0/5
Treated Dilution 5-replication N.2	0/5	0/5
Treated Dilution 5-replication N.3	0/5	0/5

Definitive test -Limit test

Control	24 hours	48 hours
Control replication N.1	0/5	0/5
Control replication N.2	0/5	0/5
Control replication N.3	0/5	0/5
Control replication N.4	0/5	0/5
Concentration 1: 100.00 mg/L
	24 hours	48 hours
Treated -replication N.1	0/5	0/5
Treated-replication N.2	0/5	0/5
Treated -replication N.3	0/5	0/5
Treated-replication N.4	0/5	0/5

Table N. 2: Concentration of dissolved oxygen (mg/L) in treated and control at the beginning and at the end of the test (48 hours) in the range finding test (at the highest tested concentration) and in the definitive test

Range finding test

Control	Beginning of test	End of test
Control replication N.1	5.97	5.00
Control replication N.2	5.74	5.01
Control replication N.3	5.76	5.01
Control replication N.4	5.99	5.00

Concentration: 1000.00 mg/L

Treated	Beginning of test	End of test
Treated replication N.1	5.67	5.07
Treated replication N.2	5.32	4.97
Treated replication N.3	5.39	4.83
Treated replication N.4	5.44	5.12

 

Definitive test - Limit test

Control	Beginning of test	End of test
Control replication N.1	6.30	5.70
Control replication N.2	6.28	5.83
Control replication N.3	6.22	5.75
Control replication N.4	6.38	5.84

Concentration: 100.00 mg/L

Treated	Be innin of test	End of test
Treatedreplication N.1	5.95	5.69
Treated replication N.2	5.90	5.74
Treated replication N.3	5.87	'5.61
Treated replication N.4	5.92	5.58

Table N 3: Concentration of the test substance at the beginning and at the end of the test (48 hours) in treated sample in the definitive test

	t=0	t=48 h	% Stability
Treated (100 mg/L)	55.02 ppm	50.71 ppm	92 stable

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 48 h EC50 for the test substance was determined to be >100 mg/L (nominal).

Executive summary:

A study was conducted to determine the acute toxicity of the test substance to Daphnia magna, according to OECD Guideline 202, in compliance with GLP. Following a range-finding test, the daphnids were exposed to 100 mg/L (nominal) of the test substance for a total period of 48 h. The test daphnids were then kept under observations for a period of 48 h and the number of immobilised organisms and/or possible abnormal behaviours both in control and in treatment group were observed. At the beginning and at the end of the test (48 h) dissolved oxygen and pH were measured in the tested concentration and in control group. Temperature of the assay environment was recorded at the beginning and at the end of the test. Total organic carbon concentration (TOC) was measured at the beginning and at the end of the test in a pool derived from control and treated vessels. All daphnids survived to the end of the test. No abnormal behaviour or appearance was observed. The test substance was considered to be stable since the TOC values at the end of the test remained within the required range (80-120%) in comparison to the initial measured values. The pH, temperature and oxygen concentration requirements for the definitive test satisfied the validity criteria. Under the study conditions, the 48 h EC50 for the test substance was determined to be >100 mg/L (nominal) (Giarei, 2020).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

A study was conducted to determine the acute toxicity of the test substance to Daphnia magna, according to OECD Guideline 202, in compliance with GLP. Following a range-finding test, the daphnids were exposed to 100 mg/L (nominal) of the test substance for a total period of 48 h. The test daphnids were then kept under observations for a period of 48 h and the number of immobilised organisms and/or possible abnormal behaviours both in control and in treatment group were observed. At the beginning and at the end of the test (48 h) dissolved oxygen and pH were measured in the tested concentration and in control group. Temperature of the assay environment was recorded at the beginning and at the end of the test. Total organic carbon concentration (TOC) was measured at the beginning and at the end of the test in a pool derived from control and treated vessels. All daphnids survived to the end of the test. No abnormal behaviour or appearance was observed. The test substance was considered to be stable since the TOC values at the end of the test remained within the required range (80-120%) in comparison to the initial measured values. The pH, temperature and oxygen concentration requirements for the definitive test satisfied the validity criteria. Under the study conditions, the 48 h EC50 for the test substance was determined to be >100 mg/L (nominal) (Giarei, 2020).