4,4'-Isopropylidenediphenol, polymer with 1-chloro-2,3-epoxypropane, propane-1,2-diol acrylate and succinic anhydride

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 29 March, 2004 to 5 May, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the ISO Standard which meets the test methods and validity criteria of the EU Method and the OECD Guideline.

Qualifier:

according to guideline

Guideline:

ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

All samples were stored frozen. On the day of analysis, the frozen samples were defrosted at room temperature. Stability of the samples under deep-freeze conditions were already determined in an another experiment. The entire volume of each sample (10 mL) was transferred quantitatively into a 20 mL volumetric flask using acetonitrile. The flasks were filled up to the mark with acetonitrile.

Vehicle:

yes

Details on test solutions:

The standard test procedures required generation of test solutions, which should contain completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that disturb the test system should be prevented (e.g. film of the test substance on the water surface). The test substance was a reddish highly viscous liquid and not completely soluble in test medium at the concentrations tested. The test substance was a mixture.

A pre-test was performed to examine the solubility of the test substance in test medium. Amounts of 103.1 and 10.41 mg of the highly viscous substance were weighed on a cover glass or on the lid of an Eppendorf cup and then added to separate volumes of 1000 mL ISO-medium. Magnetic stirring or treatment with ultrasonic waves did not result in the dissolving or dispersing of the test substance. Subsequently, acetone and methanol were tested as presolvents. Weighed amounts of 99.3 and 99.8 mg were added to 1 mL acetone and methanol, respectively. After vigorous stirring, the test substance was completely dissolved in acetone and contained undissolved test substance particles in methanol. 100 µL of the solution in acetone was added 1 L ISO-medium, resulting in a slightly hazy solution with a nominal concentration of 10 mg/L. Finally, a weighed amount of 1.0004 g was added to 1 mL acetone and vigorously stirred for 25-30 min, after which it was completely dissolved. 200 µL of this solution was added to 1 L ISO-medium, resulting in a clear solution with pink test substance droplets. Magnetic stirring overnight did not result in the dissolving or dispersing of the test substance.
In the range-finding test, preparation of test solutions of 10 mg/L and below started with stock solutions in acetone (Pestiscan 99.8%, Labscan, Ireland) at concentrations a factor of 10000 above the target concentrations in test medium. Subsequently, amounts of 500 µL were added to 5000 mL lSO medium. After a magnetic stirring period of 30 min the resulting solutions were slightly hazy (10 mg/L) or clear and colourless (1 and 0.1 mg/L). The test solutions originated from those used for the simultaneously performed acute fish toxicity test. The highest test concentration (i.e. loading rate of 100 mg/L) was prepared using a stock of 500 mg test substance in 500 mg acetone, which was added to 5 L ISO medium. Following 24 h of magnetic stirring and 3 h of stabilisation, this solution was clear but contained precipitate. Therefore, the Water Accommodated Fraction (WAF) was siphoned off and used as such. The final test solution was clear and colourless.

In the limit test, 1 g of test substance was mixed with 1 g of acetone (Pestiscan 99.8%, Labscan, Ireland) and then added to 10 L of ISO-medium. After 48 h of magnetic stirring and a 24 h stabilisation period, the solution was clear and contained test substance precipitate and a floating layer. Therefore, the WAF was siphoned off and used as such. The final test solution was clear and colourless. The test solutions originated from those used for the simultaneously performed acute fish toxicity test. Note that silanised glassware was used in the limit test to prevent possible adsorption of the test substance to glass. To this end, glassware was rinsed twice with 2% dichloromethylsilane (Acros organics 99%, Belgium) in heptane (Pestiscan 95%, Labscan, Ireland). Subsequently, heptane was evaporated under pressure air. Finally, glassware was rinsed three times with Milli-Ro water and dried in an incubator at 85°C.

Test organisms (species):

Daphnia magna

Details on test organisms:

-Source: In-house laboratory culture with a known history.
-Reason for selection: This system has been selected as an internationally accepted invertebrate species.
-Validity of batch: Daphnids originated from a healthy stock, 2 to 5 brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
-Characteristics: For the test selection of young daphnia with an age of < 24 h, from parental daphnids of more than two wk old.

Breeding:
-Start of each batch: With newborn daphnids, i.e. less than 3 d old, by placing about 250 of them into 5 L of medium in an all-glass culture vessel.
-Maximum age of the cultures: 4 wk
-Renewal of the cultures: After 7 d of cultivation half of the medium twice a wk.
-Temperature of medium: 18-22⁰C
-Feeding: Daily, a suspension of fresh water algae.
-Medium: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

250 mg/L expressed as CaCO3

Test temperature:

18-22⁰C

pH:

6-8.5

Dissolved oxygen:

≥7 mg/L at the start and ≥5 mg/L at the end

Nominal and measured concentrations:

Measured concentration (limit test): 0.86 mg/L

Details on test conditions:

A range-finding test was performed to provide information about the range of concentrations to be used in a final test. The decrease of test substance concentrations during the test period was very likely caused by the extremely low water solubility of the test substance. However, to eliminate the possibility of adsorption of the test substance to glass it was decided to perform the limit test in silanised glassware.

Limit test:
-Test concentrations
Test substance: A WAF prepared at a loading rate of 100 mg/L.
Solvent-control: Test medium containing acetone used in the treatment of the stock solutions.
-Test procedure and conditions
Test duration: 48 h
Test type: Static
Test vessels: 100 mL, all-glass
Medium: ISO
Number of daphnia: 20 per concentration
Loading: 5 per vessel containing 80 mL medium
Light: 16 h photoperiod daily
Feeding: No feeding
Aeration: No aeration of the test solutions
Introduction of daphnia: Within 3/4 hours after preparation of the test solutions.

Sampling for analysis of test concentrations: During the limit test samples were taken from the WAF prepared at a loading rate of 100 mg/L and the solvent-control for analysis.
Sampling: Frequency: at t=O hand t=48 h, Volume: 10 mL from the approximate centre of the test vessels, Storage: Samples were stored in a freezer until analysis. Additionally, reserve samples of 10 mL were taken from all test solutions 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.

Measurements and recordings: Immobility (including mortality) at 24 h and at 48 h. The pH and dissolved oxygen were recorded at the beginning and at the end of the test, for the limit concentration and the control. Temperature of medium was measured continuously in a temperature control vessel beginning at the start of the test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: based on loading rate (WAF)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

ca. 0.11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Range-finding test: After 48 h of exposure a total of 50% immobilised daphnids was observed at 10 mg/L, while only 20% was immobilized in the WAF prepared at a loading rate of 100 mg/L. Analytical results showed that this was a consequence of the actual exposure concentrations which, especially at 10 mg/L highly exceeded the maximum solubility. Initial measured concentrations were 4.9 and 0.43 mg/L at 10 mg/L and the WAF prepared at a loading rate of 100 mg/L, respectively (based on the analytical results of the simultaneously performed range-finding test with carp). Note that no visible undissolved material was observed. The 48h-ECso was expected to be above the water solubility of the test substance and the observed effects were most likely related to the fact that concentrations exceeded the solubility limit. The decrease of test substance concentrations during the test period was most likely caused by the extremely low water solubility of the test substance. However, to eliminate the possibility of adsorption of the test substance to glass it was decided to perform the limit test in silanised glassware.

Limit test:
- Measured concentrations: Analysis of the sample taken at the start (t=0) of the limit test showed a measured concentration of 0.86 mg/L, which was well above the water solubility. After 48 h of exposure the test concentration had decreased below the limit of detection (Le. below 0.03 mg/L). The average exposure concentration was calculated to be 0.11 mg/L, which was above water solubility.
- Immobility: After 48 h of exposure 35% immobility was recorded in the limit concentration, while no immobility was observed in the solvent-control. Despite the fact that the initial test concentration was (far) above the limit of solubility (i.e. < 0.007 mg/L), no visible undissolved material was observed. This was probably due to the nature of the test substance, which is a viscous liquid. However, the observed immobile daphnids were probably a consequence of the fact that the test solution was oversaturated.
- Determination of effect concentrations: Below table shows the effect parameters based on both loading rate and average exposure concentration. Based on the results of the limit test no NOEC can be given.

Parameter Loading rate of the test substance (mg/L) Average concentration of the test substance (mg/L)
48 h EC50 >100 >0.11

Results with reference substance (positive control):

The actual responses in this reference test with potassium dichromate are within the ranges of the expected responses at the different concentrations. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected.
The 24h-EC50 was 0.84 mg/L with a 95% confidence interval between 0.78 and 0.94 mg/L.
The 48h-EC50 was 0.66 mg/L with a 95% confidence interval between 0.54 and 0.87 mg/L.

Table 1. Incidence of immobility in the range-finding study:

Nominal conc. of test substance (mg/L)	Vessel number	Number Daphnia exposed	Response at 24 h		Response at 48 h
			Number	Total %	Number	Total %
Blank-control	A B	5 5	0 0	0	0 0	0
Solvent-control	A B	5 5	0 0	0	0 0	0
0.1	A B	5 5	0 0	0	0 0	0
1	A B	5 5	0 1	10	0 1	10
10	A B	5 5	0 0	0	3 2	50
100	A B	5 5	1 0	10	0 2	20

 

Table 2. Acute immobilization of daphnia after 24 and 48 h in the limit test

 

Loading rate of test substance (mg/L)	Vessel number	Number Daphnia exposed	Response at 24 h		Response at 48 h
			Number	Total %	Number	Total %
Solvent-control	A B C D	5 5 5 5	0 0 0 0	0	0 0 0 0	0
100	A B C D	5 5 5 5	0 0 0 0	0	1 2 2 2	35

 

Analytical results:

Table 3. Procedural recovery samples:

Date of preparation (dd-mm-yy)	Date of analysis (dd-mm-yy)	Concentration nominal (mg/L)	Concentration analyzed (mg/L)	Recovery (%)	Mean recovery (%)
10-May-04	10-May-04	0.0515 0.0515	No quantitative results an interfering peak was observed close to the position of the test substance
10-May-04	10-May-04	103 103	99.3 102	96 99	98

 

Table 2. Concentration of test substance in test medium (limit test):

Time of sampling (h)	Date of sampling (dd-mm-yy)	Date of analysis (dd-mm-yy)	Concentration
			Loading rate (WAF)(mg/L)	Analysed (mg/L)	Relative to initial (%)
0	03-May-04	10-May-04	0 (solvent-control) 100	Not detected 0.855	Not applicable
48	05-May-04	10-May-04	0 (solvent control) 100	Not detected <LOD (limit of detectionwas determined to be 0.03 mg/L taking the dilution factor of the samples (2) into account.

 

Validity criteria fulfilled:

yes

Remarks:

No daphnia became immobilised or trapped at the surface of the water in the control, test conditions were within the limits, positive control was valid.

Conclusions:

Under the conditions of study, the 48 h EC50 was expected to be above the water solubility of the test substance and EL50 therefore is greater than 100 mg/L, corresponding to an average exposure concentration of 0.11 mg/L.

Executive summary:

A study was conducted to assess the acute toxicity of the substance to Daphnia magna according to the ISO International Standard 6341, 1996, OECD Guideline 202 and EU Method C.2, in compliance with GLP. The batch of the test substance was a reddish highly viscous liquid and not completely soluble in test medium at the tested concentrations. The test substance was a mixture. The water solubility at 19.9 +/- 0.5°C was determined to be <7 x 10-3 mg/L, using the flask method (NOTOX project 403829). A range-finding test was performed exposing 10 daphnids per concentration to a blank-control, a solvent-control, 0.1, 1.0 and 10 mg/L and to a WAF prepared at a loading rate of 100 mg/L. After 48 h of exposure, 50% immobilised daphnids were observed at 10 mg/L, while only 20% was immobilised in the WAF prepared at a loading rate of 100 mg/L. Analysis of samples taken at the start of the test showed that the initial concentrations at nominal 10 mg/L and at a WAF prepared at a loading rate of 100 mg/L were well above the water solubility of test substance. These concentrations did not remain stable during the 48-h test period. The decrease of test substance concentrations during the test period was most likely caused by its extremely low water solubility. However, to eliminate the possibility of adsorption of the test substance to glass, a limit test was performed using silanised glassware. In the limit test, 20 daphnids per concentration were exposed to a WAF prepared at a loading rate of 100 mg/L and a solvent-control. Analysis of the sample taken at the start of the limit test showed a measured concentration of 0.86 mg/L. After 48 h of exposure the test concentration had decreased below the limit of detection (i.e. below 0.03 mg/L). The average exposure concentration was calculated to be 0.11 mg/L, which was above water solubility. The test substance only induced 35% acute immobilisation following exposure to a WAF prepared at a loading rate of 100 mg/L, which corresponds to average exposure concentration of 0.11 mg/L. Therefore, the 48 h EC50 was expected to be above the water solubility of the test substance and EL50 therefore is greater than 100 mg/L, corresponding to an average exposure concentration of 0.11 mg/L (Bouwman, 2004).

Description of key information

Based on the conditions of the study, the 48 h EC50 was expected to be above the water solubility of the test substance and EL50 therefore is greater than 100 mg/L, corresponding to an average exposure concentration of 0.11 mg/L..

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.11 mg/L

Additional information

A study was conducted to assess the acute toxicity of the substance to Daphnia magna according to the ISO International Standard 6341, 1996, OECD Guideline 202 and EU Method C.2, in compliance with GLP. The batch of the test substance was a reddish highly viscous liquid and not completely soluble in test medium at the tested concentrations. The test substance was a mixture. The water solubility at 19.9 +/- 0.5°C was determined to be <7 x 10-3 mg/L, using the flask method (NOTOX project 403829). A range-finding test was performed exposing 10 daphnids per concentration to a blank-control, a solvent-control, 0.1, 1.0 and 10 mg/L and to a WAF prepared at a loading rate of 100 mg/L. After 48 h of exposure, 50% immobilised daphnids were observed at 10 mg/L, while only 20% was immobilised in the WAF prepared at a loading rate of 100 mg/L. Analysis of samples taken at the start of the test showed that the initial concentrations at nominal 10 mg/L and at a WAF prepared at a loading rate of 100 mg/L were well above the water solubility of test substance. These concentrations did not remain stable during the 48-h test period. The decrease of test substance concentrations during the test period was most likely caused by its extremely low water solubility. However, to eliminate the possibility of adsorption of the test substance to glass, a limit test was performed using silanised glassware. In the limit test, 20 daphnids per concentration were exposed to a WAF prepared at a loading rate of 100 mg/L and a solvent-control. Analysis of the sample taken at the start of the limit test showed a measured concentration of 0.86 mg/L. After 48 h of exposure the test concentration had decreased below the limit of detection (i.e. below 0.03 mg/L). The average exposure concentration was calculated to be 0.11 mg/L, which was above water solubility. The test substance only induced 35% acute immobilisation following exposure to a WAF prepared at a loading rate of 100 mg/L, which corresponds to average exposure concentration of 0.11 mg/L. Therefore, the 48 h EC50 was expected to be above the water solubility of the test substance and EL50 therefore is greater than 100 mg/L, corresponding to an average exposure concentration of 0.11 mg/L (Bouwman, 2004).