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Diss Factsheets

Ecotoxicological information

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:
26 March 2018 - 22 May 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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
3-aminomethyl-3,5,5-trimethylcyclohexylamine
EC Number:
220-666-8
EC Name:
3-aminomethyl-3,5,5-trimethylcyclohexylamine
Cas Number:
2855-13-2
Molecular formula:
C10H22N2
IUPAC Name:
3-(aminomethyl)-3,5,5-trimethylcyclohexanamine
Constituent 2
Chemical structure
Reference substance name:
4-tert-butylphenol
EC Number:
202-679-0
EC Name:
4-tert-butylphenol
Cas Number:
98-54-4
Molecular formula:
C10H14O
IUPAC Name:
4-tert-butylphenol
Constituent 3
Chemical structure
Reference substance name:
2-(((5-amino-1,3,3-trimethylcyclohexyl)methylamino)methyl)-4-tert-butylphenol
Molecular formula:
C21H36N2O
IUPAC Name:
2-(((5-amino-1,3,3-trimethylcyclohexyl)methylamino)methyl)-4-tert-butylphenol
Test material form:
liquid: viscous

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control, 10, 18, 32, 56 and 100 mg/L loading rate WAF
- Sample storage conditions before analysis: The samples were stored frozen prior to analysis.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Nominal amounts of test item (50, 90, 160, 280 and 500 mg) were each separately added to the surface of 5 liters of test water to give the 10, 18, 32, 56 and 100 mg/L loading rates respectively. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1-Hour. Microscopic observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2 to 4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 10, 18, 32, 56 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed no microdispersions of test item present.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 48 hours.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: clone 5, originally supplied by University of Sheffield/UK
- Source: in-house laboratory cultures
- Feeding during test: no

ACCLIMATION
- Acclimation conditions: Elendt M7 medium
- Type and amount of food: Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h

Test conditions

Test temperature:
20 °C to 22 °C
pH:
8.1 - 9.2 at test start, 7.7-8.1 at test end
Dissolved oxygen:
8.6-8.7 mg O2/L at test start, 8.8 - 8.9 mg O2/L at test end
Nominal and measured concentrations:
10, 18, 32, 56 and 100 mg/L loading rate WAF
Details on test conditions:
TEST SYSTEM
- Test vessel: 150 mL beakers containing approximately 100 mL of test preparation
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light / 8 h darkness with 20 minute dawn and dusk transition periods
- Light intensity: 675 to 740 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure

RANGE-FINDING STUDY
- Test concentrations: Control, 0.010, 0.10, 1.0, 10 and 100 mg/L loading rate WAF
- Results used to determine the conditions for the definitive study:
No sub-lethal effects of exposure were observed throughout the test.
No immobilization was observed at 0.010, 1.0 and 10 mg/L loading rate WAF, however, immobilization was observed at 0.10 and 100 mg/L loading rate WAF.
Based on this information loading rates of 10, 18, 32, 56 and 100 mg/L were selected for the definitive test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrations
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
21 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
mortality
Remarks on result:
other: 95% Confidence limits
Remarks:
19-24 mg/L loading rate WAF
Details on results:
- Other biological observations: No sub-lethal effects of exposure were observed throughout the test at 10 mg/L loading rate WAF.

Sub-lethal effects of exposure were observed in the 18 mg/L loading rate WAF. These responses were reduced mobility and pale appearance.
- Mortality of control: none of the control daphnids showed immobilization or other signs of disease or stress

- Effect concentrations exceeding solubility of substance in test medium:
At the start of the mixing period the 10, 18 and 32 mg/L loading rates were observed to be clear colorless water columns with tiny globules of test item on surface whilst the 56 and 100 mg/L loading rates were observed to be clear colorless water columns with large and small globules of test item on surface. After 23 hours stirring and a 1-Hour standing period the 10, 18 and 32 mg/L loading rates were observed to be clear colourless water columns with small globules of test item on the surface. The 56 and 100 mg/L loading rates were observed to be clear colourless water columns with globules of test item on the surface.
Microscopic examination of the WAFs showed micro-dispersions of test item present and therefore it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2 to 4 cm in length). Microscopic examination after filtering showed the glass wool plug had removed all the undissolved test item. After siphoning and for the duration of the test, all loading rates were observed to be clear, colorless solutions.

Results with reference substance (positive control):
- Results with reference substance valid? yes
24 h EC50 = 0.79 mg/L, 95% c.l. 0.73-0.86 mg/L
48 h EC50 = 0.75 mg/L, 95% c.i. 0.56-1.0 mg/L

The results from the positive control with potassium dichromate were within the normal range for this reference item. The results of the inter laboratory tests and a Technical Corrigendum to ISO 6341 give a 24-Hour EC50 for potassium dichromate within the range 0.6 mg/L to 2.1 mg/L.
Reported statistics and error estimates:
The EL50 values and associated confidence limits at 24 hours were calculated by the binomial method. The EL50 values and associated confidence limits at 48 hours were calculated using the Trimmed Spearman-Karber method. The Lowest Observed Effect Loading Rate (LOEL) and the No Observed Effect Loading Rate (NOEL) at 24 and 48 hours were calculated using the Fisher’s Exact Binomial Test with Bonferroni correction. All results were calculated using the ToxRat Professional computer software package (TOXRAT).

Any other information on results incl. tables

Validity criteria:

The test was considered to be valid given that none of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.

Cumulative Immobilization Data and Observations in the Definitive Test 

Nominal Loading Rate (mg/L)

24 Hours

Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)

Observations

Rep1

Rep2

Rep3

Rep4

Total

%

Rep1

Rep2

Rep3

Rep4

Control

0

0

0

0

0

0

5 N

5 N

5 N

5 N

10

0

0

0

0

0

0

5 N

5 N

5 N

5 N

18

0

0

0

0

0

0

1 R 4 N

3 R 2 N

2 R 3 N

2 R 3 N

32

5

5

5

5

20

100

AI

AI

AI

AI

56

5

5

5

5

20

100

AI

AI

AI

AI

100

5

5

5

5

20

100

AI

AI

AI

AI

Nominal Loading Rate (mg/L)

48 Hours

Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)

Observations

Rep1

Rep2

Rep3

Rep4

Total

%

Rep1

Rep2

Rep3

Rep4

Control

0

0

0

0

0

0

5 N

5 N

5 N

5 N

10

0

0

0

0

0

0

5 N

5 N

5 N

5 N

18

1

1

0

2

4

20

1 P 1 R 2 N

2 P 2 R

2 P 3 R

2 P 1 R

32

5

5

5

5

20

100

AI

AI

AI

AI

56

5

5

5

5

20

100

AI

AI

AI

AI

100

5

5

5

5

20

100

AI

AI

AI

AI

Rep = Replicate

N = Normal

P = Pale

R = Reduced mobility

AI = All daphnia immobilized

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Exposure of the freshwater invertebrate Daphnia magna to the test item has been investigated and gave the following results: 48 h EL50 = 21 mg/L loading rate WAF (95% Confidence Limits 19-24 mg/L loading rate WAF).
Executive summary:

This study was performed to assess the acute toxicity of Mannich base IPDA-PTBP to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, 20 daphnids (4 replicates of 5 animals) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 10, 18, 32, 56 and 100 mg/L for 48 hours at a temperature of 20 °C to 22 °C under static test conditions. The number of immobilized daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

Chemical analysis of the fresh test preparations at 0 hours showed measured test concentrations to range from 4.8 to 24 mg/L. Chemical analysis of the aged test preparations at 48 hours showed measured test concentrations to range from 3.8 to 19 mg/L.

The dissolved test item may have been one or several components of the test item. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

Exposure of Daphnia magna to the test item gave the following results: 48 h EL50 = 21 mg/L loading rate WAF (95% Confidence Limits 19-24 mg/L loading rate WAF).