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

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

Study period:

From 29 March, 2004 to 7 May, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the ISO standards which meets the test methods and validity criteria of the EU Methods and the OECD Guidelines.

Qualifier:

according to guideline

Guideline:

ISO 7346-1 (Determination of the Acute Lethal Toxicity of Substances to a Freshwater Fish [Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)] - Part 1: Static Method)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

During the limit test, samples were taken from the water accomodated fraction (WAF) prepared at a loading rate of 100 mg/L and the solvent- control for analysis. 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 was determined in an 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. If necessary, the solutions were further diluted with 50/50 (v/v) acetonitrile/ISO-medium to obtain concentrations within the calibration range.

Vehicle:

yes

Details on test solutions:

The standard test procedures required generation of test solutions that contain completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that disturbed the test system was 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 µm of the solution in acetone was added to 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 10,000 above the target concentrations in test medium. Subsequently, amounts of 500 µm were added to 5000 mL ISO-medium. After a magnetic stirring period of 30 min the resulting solutions were slightly hazy (10 mg/L) or clear and colourless (1.0 and 0.1 mg/L). Part of the test solutions was used in the simultaneously performed acute toxicity test with Daphnia magna. 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 Water Accommodated Fraction was siphoned off and used as such. The final test solution was clear and colourless. Part of the test solutions was used in the simultaneously performed acute toxicity test with Daphnia magna. 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):

Cyprinus carpio

Details on test organisms:

-Test System
Source: Zodiac, proefacc, "De Haar Vissen", L.U. Wageningen, the Netherlands.
Mean length: Range-finding test: 2.2 ± 0.1 cm; Limit test: 2.3 ± 0.1 cm
Mean weight: Range-finding test: 0.26 ± 0.04 g; Limit test: 0.31 ± 0.07 g
Characteristics: F1 from a single parent-pair bred in UV-treated water
Reason for selection: This system has been selected as an internationally accepted species.
Total fish used: 26

-Holding
Quarantine/Acclimatisation: At least 12 d after delivery.
Medium: ISO-medium, formulated using Milli-Ro water (tapwater purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)
Measurements: pH, nitrate and nitrite concentration and ammonia concentration: once a wk. Temperature: every day.
Feeding: Daily with Trouvit.
Validity of batch: In the batch of fish used for the test, mortality during the 7 d prior to the start of the test was less than 5%.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

250 mg/L CaCO3

Test temperature:

20-24°C

pH:

6.0-8.5

Dissolved oxygen:

6.3-9.1 mg/L

Nominal and measured concentrations:

Nominal concentrations: 0 and 100 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: 96 h
Test type: Static
Test vessels: 100 mL, all-glass, containing 9.5 L of test medium
Medium: ISO-medium, aerated until the dissolved oxygen concentration had reached saturation and the pH had stabilised. After aeration the hardness was 250 mg CaCO3 per liter and the pH was 7.8.
Number of fishes: 7 fish per concentration and control
Loading: 0.23 g fish/L, i.e. 7 fish per 9.5 L of test medium
Light: 16 h photoperiod daily
Feeding: No feeding
Aeration: Aeration was introduced after 48 h of exposure and was maintained for 27 h.
Introduction of fish: Within 30 mins 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.
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=0 h, t=48 h and t=96 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 3 mo after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Measurements and recordings: Mortality and other effects at 21/2, 24, 48, 72 and 96 h following the start of exposure. Ten fish of the batch used for the test, were weighed and measured prior to the start of the test. Dissolved oxygen content, pH and temperature were measured daily.

Reference substance (positive control):

yes

Remarks:

Pentachlorophenol (PCP)

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: based on loading rate (WAF)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.082 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

-Range-finding test:

Mortality and other effects: No mortality or any other effects were observed at any of the test concentrations during the 96 h exposure period. The LC50 was expected to be above the water solubility of the test substance.

Stability of the test substance under test conditions: Analysis of the samples taken at the start (t=0) of the range-finding test showed initial concentrations of 4.9 mg/L (49%) and 0.43 mg/L (0.43%) at the nominal 10 mg/L concentration
and the WAF prepared at a loading rate of 100 mg/L, respectively. Therefore, both initial concentrations were well above the water solubility of the test substance (i.e. <0.007 mg/L). These test concentrations did not remain stable during the 96-h test period. 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 24 h of exposure the test concentration had decreased to 0.065 mg/L and further to below the limit of detection (i.e. below 0.03 mg/L) after 96 h of exposure. The average exposure concentration was calculated to be 0.082 mg/L, which was above water solubility.

Mortality and other effects: The responses recorded in the WAF prepared at a loading rate of 100 mg/L were in agreement with the results of the range-finding test.

Determination of effect concentrations: Below table shows the effect parameters based on both loading rate and average exposure concentration:

Parameter Loading rate of the test substance (mg/L) Average concentration of the test substance (mg/L)
NOEC 100 0.082
96 h LC50 >100 >0.082

Results with reference substance (positive control):

In the reference test, PCP induced no lethal effects in carp at or below 0.22 mg/L. The 96 h LC50 for carp exposed to PCP was 0.32 mg/: (95 % confidence interval between 0.22 and 0.46 mg/L) and already reached within 24 h of exposure. The range of the 96 h LC50 for carp is generally between 0.10 and 0.46 mg/L based on historical data of reference tests performed approx every 3 mo from April 1988 until the end 2000, and annually since then. The response observed in carp originating from the present batch falls within this range.

Sublethal observations / clinical signs:

Table 1. Incidence ofmortality and total mortality in the range-finding study:

Nominal conc. of test substance (mg/L)	Initial number of fish	Cumulative responsibility					Total mortality (%)
		2 h	24 h	48 h	72 h	96 h
0.1	3	0	0	0	0	0	0
1	3	0	0	0	0	0	0
10	3	0	0	0	0	0	0
100	3	0	0	0	0	0	0

 

Table 2. Incidence of mortality and total mortality during the limit test:

 

Loading rate of test substance (mg/L)	Average concentration of the test substance (mg/L)	Initial number of fish	Cumulative mortality					Total mortality (%)
			2 h and 30 min	24 h	48 h	72 h	96 h
Solvent-control	Solvent-control	7	0	0	0	0	0	0
100	0.082	7	0	0	0	0	0	0

 

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 (%)
7-Apr-04	7-Apr-04	0.0996 0.0996	0.0918 0.0934	92 94	93
7-Apr-04	7-Apr-04	100 100	101 101
10-May-04	10-May-04	0.0515 0.0515	In these samples an interfering peak was observed close to the position of the test substance. Therefore no quantitative results could be obtained for these samples.	101 101	101
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
			Nominal (mg/L)	Analysed (mg/L)	Relative to nominal (%)	Relative to initial (%)
0	29-March-04	07-April-04	10	4.93	49	53
24	30-March-04	07-April-04	10	2.64	26
96	02-April-04	07-April-04	10	<LOD	N.A	N.A

 

Table 3. Concentration of test substance in test medium (range-finding study):

Time of sampling (h)	Date of sampling (dd-mm-yy)	Date of analysis (dd-mm-yy)	Concentration
			Loading rate (mg/L)	Analyzed (mg/L)	Relative to initial (%)
0	29-March-04	07-April-04	100	0.430
24	30-March-04	07-April-04	100	<LOD	N.A
96	02-April-04	07-April-04	100	<LOD	N.A

 

Table 4. 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)	Analyzed (mg/L)	Relative to initial (%)
0	3-May-04	10-May-04	0(solvent-control) 100	<LOD 0.855	8
24	4-May-04	10-May-04	0 100	<LOD 0.0651
96	7-May-04	10-May-04	0 100	<LOD <LOD

 

Validity criteria fulfilled:

yes

Remarks:

No mortality in control; constant condition; dissolved O2 concentration > 60 % of saturation value; valid reference and analytical results

Conclusions:

Under the conditions of the study, the 96 h LC50 was expected to be above the water solubility of the test substance and LL50 therefore is greater than 100 mg/L, corresponding to an average exposure concentration of 0.082 mg/L.

Executive summary:

A study was conducted to assess the acute toxicity of the substance to common carp (Cyprinus carpio) according to the ISO International Standard 7346 -1, EU Method C.1 and OECD Guideline 203, 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 three fish per concentration to 0.1, 1.0 and 10 mg/Land to a water accommodated fraction (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 96-h test period. No mortality or other effects were observed throughout the study. The LC50 was expected to be above the water solubility of the test substance and the LL50 therefore is greater than 100 mg/L under the tested conditions. 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, 7 fish per concentration were exposed to a WAF prepared at a loading rate of 100 mg/L and a solvent control. The total test period was 96 h. Analysis of the sample taken at the start of the limit test showed a measured concentration of 0.86 mg/L. After 24 h of exposure the test concentration had decreased to 0.065 mg/Land further to below the limit of detection (i.e. below 0.03 mg/L) after 96 h. The average exposure concentration was calculated to be 0.082 mg/L, which was above water solubility. Under the conditions of the study, concentration levels that might be toxic for carp could not be reached because of the extremely low solubility of the test substance in water. Hence, the LC50 was expected to be above the water solubility of the test substance and LL50 therefore is greater than 100 mg/L, corresponding to an average exposure concentration of 0.082 mg/L (Bouwman, 2004).

Description of key information

Based on the conditions of the study, the 96 h LC50 was expected to be above the water solubility of the test substance and LL50 therefore is greater than 100 mg/L, corresponding to an average exposure concentration of 0.082 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.082 mg/L

Additional information

A study was conducted to assess the acute toxicity of the substance to common carp (Cyprinus carpio) according to the ISO International Standard 7346 -1, EU Method C.1 and OECD Guideline 203, 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 three fish per concentration to 0.1, 1.0 and 10 mg/Land to a water accommodated fraction (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 96-h test period. No mortality or other effects were observed throughout the study. The LC50 was expected to be above the water solubility of the test substance and the LL50 therefore is greater than 100 mg/L under the tested conditions. 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, 7 fish per concentration were exposed to a WAF prepared at a loading rate of 100 mg/L and a solvent control. The total test period was 96 h. Analysis of the sample taken at the start of the limit test showed a measured concentration of 0.86 mg/L. After 24 h of exposure the test concentration had decreased to 0.065 mg/Land further to below the limit of detection (i.e. below 0.03 mg/L) after 96 h. The average exposure concentration was calculated to be 0.082 mg/L, which was above water solubility. Under the conditions of the study, concentration levels that might be toxic for carp could not be reached because of the extremely low solubility of the test substance in water. Hence, the LC50 was expected to be above the water solubility of the test substance and LL50 therefore is greater than 100 mg/L, corresponding to an average exposure concentration of 0.082 mg/L (Bouwman, 2004).