Reaction products of 4,4'-methylenebis(cyclohexylamine) and 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

During the final test samples for analysis were taken from the control, 3.2, 10 and 32 mgll at the
start of the test. After 24 hours a sample was taken from 18 mgll. At the end of the test period
samples for analysis were taken from 10 mg/l and the control.
Sampling:

Volume - 10 or 20 ml from the approximate centre of the test
vessels.
Storage - Samples not analysed on the day of sampling were stored in a freezer until analysis.
Additionally, reserve samples of 10 or 20 ml were taken from all test solutions (with surviving
fish) for possible analysis. If not already used, these samples were stored in a freezer for a
maximum of three months after delivery of the draft report, pending on the decision of the
sponsor for additional analysis. The method of analysis and specification of the samples
analysed are described in the appended Analytical Report.

Details on test solutions:

Medium Tap-water

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Species
Source
Mean length
Mean weight
Characteristics
Reason for selection
Total fish used
Rainbow trout (Oncorhynchus mykiss, Walbaum
1988)
Trout Hatchery Blitterswijk, the Netherlands.
First range-finding test: 4.8 * 0.51 cm
Second range-finding test: 3.97 * 0.1 6 cm
First LC50 test: 4.1 1 * 0.17 cm
Second LCs0 test: 4.6 rt 0.31 cm
Final LCs0 test: 5.51 * 0.57 cm
First range-finding test: 1.82 * 0.67 g
Second range-finding test: 0.90 + 0.08 g
First LCa test: 1.32 * 0.23 g
Second test: 1.66 + 0.33 g
Final LC50 test: 2.24 + 0.48 g
F1 from a single parent-pair bred in UV-treated water
This system has been selected as an internationally
accepted species.
Total fish used - 181

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

MEASUREMENTS AND RECORDINGS

Mortality and other effects
Fish length and weight
At 3, 24,48, 72 and 96 hours following the start of
exposure. Dead fish were removed when observed.
Ten fish of the batch used for the test, were weighed
and measured prior to the start of the test.
Dissolved oxygen content and pH
Temperature of medium
Daily in all vessels, beginning at the start of the test (day 0).
Daily in one control vessel, beginning at the start of the test.

Hardness:

2.6 mmol/l

Test temperature:

22 C

Nominal and measured concentrations:

Concentrations of PACM BADGE POLYMER ADDUCT in test medium (final LC50 test).

Nominal (mg/l) Measured(mg/l)
3.2 2.76 (t=0)
10.0 8.16 (t=0)
32.0 27.7 (t=0)

18.0 7.25 (t=24)

10.0 4.04 (t=96)

Details on test conditions:

Test vessels 40 litres, all-glass.
Test medium Tap-water (data of analysis are attached).
Number of fish 7 fish per concentration and control.
Loading 0.52 g fishllitre, i.e. 7 fish per 30 litres of test medium.
Illumination 16 hours photoperiod daily.
Aeration The test media were aerated continuously during the
test.
Feeding No feeding from 48 hours prior to the test and during
the total test period.
Introduction of fish Directly after preparation of the test media.
Euthanasia At the end of the test the surviving fish were rapidly killed by exposing them to ca. 1.2% ethylene glycol monophenylether in water.

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

5.8 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

3 h

Dose descriptor:

LC50

Effect conc.:

24 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

13 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

First LC50 study:
Preparation procedures during the first study were based on the second range-finding test, which showed detectable concentrations. The only difference was the stirring period that was
prolonged to three days.
However, unexpectedly, analysis of the samples taken from all concentrations at the start of the study showed that measured concentrations were all below the limit of detection limit.
The biological results showed further no mortality up to and including the highest tested concentration, being a WAF prepared at 32 mg/l
It was obvious that due to the character of the test substance (viscous, sticky and extremely low soluble in the neutral pH-range) it was difficult to obtain detectable test concentrations and it
was also difficult to repeatably prepare the same actual exposure concentrations.
As a consequence, another solubility pre-test was performed. A stock solution was prepared at 1000 mg/l in RO-water with a pH adjusted to 2. This stock solution could completely be
dissolved after magnetic stirring and ultrasonic treatment. Test solutions were subsequently prepared by diluting this stock in tap-water and pH levels were re-adjusted to 6.0 before
introduction of the fish. A pH of 6.0 is the lowest pH level that does not affect the fish (optimum pH range: 6.0-9.0).

Second LC50 study:
All fish exposed to nominal concentrations of 18 and 32 mgA died during the first 24-hour test period, while another 4 fish exposed to nominally 10 mgA had died at the end of the test period.
No mortality or visible effects were observed in the lower test concentrations 3.2 and 5.6 mgll. The pH was maintained within 6.0 to 6.8 and the oxygen concentrations were between 9.7 and 10.5 mg/l during the test
period. Temperature was between 15.4 and 1 6.0°C.
Unfortunately, analyses of the samples taken at the start of the study were not successful. This was a consequence of the use of orthophosphoric acid used to adjust the pH of the test
solutions to 6.0 before introduction of the fish. Orthophosphoric acid disturbed the spectrophotometric measurements.
The study was repeated and this time only hydrochloric acid was used to adjust the pH of the test solutions.

Final LC50 study:
Analysis of the samples taken during the final test showed that the measured concentrations in the samples taken at the start of the test were 2.76 mg/l (86%), 8.16 mg/l (82%) and 27.7 mg/l
(87%) at the nominal concentrations of 3.2, 10 and 32 mg/l, respectively. After 24 hours of exposure the measured concentration at nominally 18 mg/l was 7.25 mg/l. The start
concentration at 18 mg/l was expected to be approximately 15.3 mg/l based on a recovery of 85%, which was the average recovery in the samples analysed at the start of exposure.
The measured concentration at 10 mg/l had decreased to 4.08 mg/l at the end of the test period.
The observed decrease in measured concentrations was almost certainly related to the fact that the pH-values of the test solutions increased from 6.0 to approximately 8.0 during the test
period. This was probably due to the fact that test solutions were continuously aerated, which resulted in a decreased solubility of PACM BADGE POLYMER ADDUCT in the test medium
due to the pH rise. This could not be prevented as the use of buffers to keep pH values more stable (like orthophosphoric acid) resulted in disturbance of the analytical determination of the
actual exposure concentrations.
The average exposure concentrations in the test solutions essential for determination of the 24-hour toxicity parameters were calculated to be 5.8 and 10.5 mgll, at the nominal concentrations
10 and 18 mgA, respectively. The toxicity parameters after 3 hours of exposure were calculated based on the (expected) concentrations measured at the start of exposure.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present test PACM BADGE POLYMER ADDUCT induced no lethal effects in Rainbow trout at or below nominally 10 mg/l, corresponding with an average exposure concentration of 5.8 mg/l.
The 3h-LC50, for rainbow trout exposed to PACM BADGE POLYMER ADDUCT based on nominal concentrations equaled 24 mg/l. Based on initial measured exposure concentrations this corresponded with 21 mg/l.
The 96h-LC50, for rainbow trout exposed to PACM BADGE POLYMER ADDUCT based on nominal concentrations was already reached after 24 hours of exposure and equalled 13 mg/l.

Executive summary:

Under the conditions of the present test PACM BADGE POLYMER ADDUCT induced no lethal effects in Rainbow trout at or below nominally 10 mg/l, corresponding with an average exposure concentration of 5.8 mg/l.

The 3h-LC50, for rainbow trout exposed to PACM BADGE POLYMER ADDUCT based on nominal concentrations equaled 24 mg/l. Based on initial measured exposure concentrations this corresponded with 21 mg/l.

The 96h-LC50, for rainbow trout exposed to PACM BADGE POLYMER ADDUCT based on nominal concentrations was already reached after 24 hours of exposure and equalled 13 mg/l.

Description of key information

Under the conditions of the present test PACM BADGE POLYMER ADDUCT induced no lethal effects in Rainbow trout at or below nominally 10 mg/l, corresponding with an average exposure concentration of 5.8 mg/l.

The 3h-LC50, for rainbow trout exposed to PACM BADGE POLYMER ADDUCT based on nominal concentrations equalled 24 mg/l. Based on initial measured exposure concentrations this corresponded with 21 mg/l.

The 96h-LC50, for rainbow trout exposed to PACM BADGE POLYMER ADDUCT based on nominal concentrations was already reached after 24 hours of exposure and equalled 13 mg/l.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

13 mg/L