N-(1,3-dimethylbutylidene)-5-[(1,3-dimethylbutylidene)amino]-1,3,3-trimethylcyclohexanemethylamine

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:

23/01/2020-17/09/2020

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)

GLP compliance:

yes

Specific details on test material used for the study:

Information as provided by the Sponsor. A Certificate of Analysis supplied by the Sponsor is
given in Annex 1.
Identification: CAS 71077-09-3
Batch: 12846
CAS Number: 71077-09-3
Purity: 97.32%
Physical State/Appearance: Pale yellow viscous liquid
Expiry Date: 30 November 2020
Storage Conditions: Room temperature in the dark, under nitrogen

Analytical monitoring:

not specified

Vehicle:

no

Details on test solutions:

Based on the results of the range-finding test the following loading rates were assigned to the
definitive test: 10, 18, 32, 56 and 100 mg/L.
Nominal amounts of test item (20, 36, 64, 112 and 200 mg) were each separately added to the
surface of 2.0 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. Visual
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 to remove the undissolved test item from the test
solutions. A 10 and 2.0 mL pipette, covered at one end with Parafilm was submerged into the
vessels, sealed end down to mid-depth within the vessels. A length of Tygon tubing was
attached to the 10 mL pipette and the Parafilm was removed using the 2.0 ml pipette. 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 micro-dispersions of test item to be present.
The concentration and stability of the test item in the test preparations were verified by
chemical analysis at 0 and 48 hours (see Annex 5).

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using first instar Daphnia magna derived from in-house laboratory
cultures.
Adult daphnids were maintained in 150 mL glass vessels containing 100 mL Elendt M4
medium (see Annex 2) in a temperature controlled room maintaining the water temperature at
18 to 22 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness
cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a
mixture of algal suspension (Raphidocelis subcapitata) and GEMMA Micro 300 fish food
suspension. Culture conditions ensured that reproduction was by parthenogenesis. 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.
A positive control test using potassium dichromate as the reference item was performed twice
in a 12 month period to demonstrate satisfactory conditions of the test. Details of the most
recent positive control (Covance study number GV41TT) are given in Annex 3.

Test type:

flow-through

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Reference substance (positive control):

not specified

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

ca. 20 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

6.1 Range-finding Test
Cumulative immobilization data and other observations from the exposure of Daphnia magna
to the test item during the range-finding test are given in Table 1.
No sub-lethal effects of exposure were observed throughout the test.
No immobilization was observed at 1.0 and 10 mg/L loading rate WAF, however,
immobilization was observed at 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.
Information provided by the Sponsor indicated that the test item degrades after approximately
2 hours and therefore the chemical analysis was based on one of the degradant products that
were determined.
Chemical analysis of the test preparations at 0 hours (see Annex 5) showed measured test
concentrations of Isophoronediamine to range from 0.61 to 50 mg/L. There was no
significant change in the measured concentrations at 48 hours indicating that the degradant
product being measured was stable under test conditions.
Cumulative immobilization data and other observations from the exposure of Daphnia magna
to the test item during the definitive test are given in Table 2. The relationship between
percentage immobilization and concentration at 48 hours for nominal loading rates is given in
Figure 1. The relationship between percentage immobilization and concentration at 48 hours
for 0-Hour measured test concentrations is given in Figure 2.
Analysis of the immobilization data by Weibull analysis using Linear Maximum-Likelihood
regression method at 24 and 48 hours based on the nominal loading rates gave the following
results:
Time
(Hour)
EL50
(mg/L Loading Rate)
95% Confidence limits
(mg/L Loading Rate WAF)
24 98 22 - 442
48 46 18 - 118
The NOEL rates after 24 and 48 hours exposure were 56 and 32 mg/L loading rate WAFs
respectively. Correspondingly the LOEL rates were considered to be 100 and 56 mg/L
loading rate WAFs.
The slopes and their standard errors of the response curves at 24 and 48 hours were 6.8
(standard error = 0.052) and 10 (standard error = 0.024) respectively.
Analysis of the immobilization data by Weibull analysis using Linear Maximum-Likelihood
regression method at 24 and 48 hours based on the 0-Hour measured test concentrations gave
the following results:
Time
(Hour)
EL50
(mg/L)
95% Confidence limits
(mg/L)
24 43 9.7 - 190
48 20 7.3 - 55
The NOEL rates after 24 and 48 hours exposure were 24 and 13 mg/L loading rate WAFs
respectively. Correspondingly the LOEL rates were considered to be 44 and 24 mg/L loading
rate WAFs.
The slopes and their standard errors of the response curves at 24 and 48 hours were 6.9
(standard error = 0.050) and 11 (standard error = 0.023) respectively.
Sub-lethal effects of exposure were observed in 18, 56 and 100 mg/L loading rate test groups.
These responses were reduced mobility (see Table 2).

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Daphnia magna based on nominal loading rates gave the following results:
Time Point
(Hours)
EL50
(mg/L Loading
Rate WAF)
95% Confidence Limits
(mg/L Loading Rate
WAF)
NOEL
(mg/L Loading
rate WAF)
LOEL
(mg/L Loading
Rate WAF)
48 46 18 - 118 32 56
Exposure of Daphnia magna based on 0-Hour measured concentrations gave the following
results:
Time Point
(Hours)
EL50
(mg/L)
95% Confidence Limits
(mg/L)
NOEL
(mg/L)
LOEL
(mg/L)
48 20 7.3 - 55 13 24

Executive summary:

1 SUMMARY

1.1 Introduction

A study was performed to assess the acute toxicity of the test item 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.

1.2 Methods

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 21 °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.

1.3 Results

Information provided by the Sponsor indicated that the test item degraded within 2 hours and

as a result of the method of preparation for the test samples, chemical analysis was performed

on a degradant product instead of the parent material. The chemical analysis was performed

on Isophoronediamine, one of identified degradant products of the parent material.

Chemical analysis of the fresh test preparations at 0 hours showed measured test

concentrations of Isophoronediamine to range from 4.5 to 44 mg/L. Chemical analysis of the

aged test preparations at 48 hours showed measured test concentrations of Isophoronediamine

to range from 4.6 to 43 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 and at the request of the

Sponsor results were also based on the 0-Hour measured concentrations of

Isophoronediamine.

Exposure of Daphnia magna based on nominal loading rates gave the following results:

Time Point

(Hours)

EL50

(mg/L Loading

Rate WAF)

95% Confidence Limits

(mg/L Loading Rate

WAF)

NOEL

(mg/L Loading

Rate WAF)

LOEL

(mg/L Loading

Rate WAF)

48 46 18 - 118 32 56

Exposure of Daphnia magna based on 0-Hour measured concentrations gave the following

results:

Time Point

(Hours)

EL50

(mg/L)

95% Confidence Limits

(mg/L)

NOEL

(mg/L)

LOEL

(mg/L)

48 20 7.3 - 55 13 24

NOEL =

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

20 mg/L