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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 29 March, 2004 to 6 May, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the ISO 8692 and methods similar to OECD Guideline 201 and EU Method C.3, in compliance with GLP.

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Deviations:

yes

Remarks:

There was a deviation in the pH, however this did not affect the integrity of the study.

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Frequency: At t=0 h, t=24 hand t=72 h
Volume: 10 mL
Storage: Samples were stored in a freezer until analysis.
Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at the limit concentration but without algae and samples for analysis were taken at the start, after 24 h of exposure and at the end of the test period. 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.

Vehicle:

yes

Details on test solutions:

The standard test procedures required generation of test solutions, which contained completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that would disturb the test system was prevented as much as possible (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 a 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 to 1 liter 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 liter 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 at concentrations a factor of 10,000 above the target concentrations in test medium. Subsequently, amounts of 50 µL were added to 500 mL M2-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). The highest test concentration (i.e. loading rate of 100 mg/L) was prepared using a stock of 100 mg test substance in 100 mg acetone, which was added to 1 liter M2-medium. Following 24 h of magnetic stirring and 2 1/4 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, 100 mg of test substance was mixed with 100 mg of acetone and then added to 1 liter of M2-medium. After 48 hours 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. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL. 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 in heptane. 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):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture.
-Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 23 ± 2°C.
-Light intensity: 60 to 120 µE/m2/s when measured in the photo synthetically effective wavelength range of 400 to 700 nm.
-Stock culture medium: M1; formulated using Milli-Ro water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)
-Pre-culture: 3 d before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 2.104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
-Pre-culture medium: M2; according to the ISO-Standard "Algal growth inhibition test" Nov. 1989; formulated using Milli-Q water (tap water purified by reverse osmosis (milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges: Milli-Q water; Millipore Corp., Bedford, Mass., USA) preventing precipitation.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

23.9 and 24.3°C

pH:

8.1-10.3

Nominal and measured concentrations:

Nominal concentrations for range-finding test: 0.1, 1.0 and 10 mg/L
Average exposure concentration for limit test: 0.068 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.
Limit test
-Test concentrations
Test substance: A WAF prepared at a loading rate of 100 mg/L
Controls: Test medium without test substance or other additives (blank-control) and test medium containing acetone used in the treatment of the stock solutions (solvent control).
Replicates: 6 replicates of the test concentration; 6 replicates of both control groups; 1 replicate of the test concentration without algae; 1 extra replicate of each solution for analysis.
-Test procedures and conditions
Test duration: 72 h
Test type: Static
Test vessels: 100 mL, all-glass, containing 50 mL of test medium
Medium: M2
Cell density: An initial cell density of 1 x 104 cells/mL.
Illumination: Continuously using TLD-Iamps of the type 'Cool-white' of 30 Watt, with a light intensity within the range of 75 to 104 µE.m-2 .s-1.
Incubation: Vessels were distributed at random in the incubator. During incubation the algal cells were kept in suspension by continuous shaking.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Cell growth inhibition and growth rate reduction

Remarks on result:

other: based on loading rate (WAF)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.068 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: cell growth inhibition and growth rate reduction

Details on results:

Range-finding test:
- Mean cell densities, inhibition of cell growth and reduction of growth rate: The mean cell densities measured during the range-finding test are presented in Table 1. Table 2 presents the percentages growth inhibition and growth rate reduction per concentration. Note that after 72 h of exposure particles in the solutions and a white layer attached to the glass just above the surface of the test solutions were observed in the solvent-control and all test concentrations. This was probably related to bacterial growth due to the acetone used in the preparation of test solutions. The results showed that both the EC50 for cell growth inhibition and growth rate reduction were 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 7.8 mg/L (78%) and 0.84 mg/L (0.84%) 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 (Le. <0.007 mg/L). These measured test concentrations did not remain stable during the 72 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.
-Experimental conditions: Only for the highest test concentration the pH was within the limits prescribed by the protocol (6.0-9.0, preferably not varying by more than 1.5 unit). The temperature of the test medium was 22.9°C at the start of the test. During the exposure period the temperature in the incubator was maintained between 23.9 and 24.3°C. Temperature remained within the limits prescribed by the protocol (21-25°C, constant within 2°C).

Limit test:
- Measured test substance concentrations: Analysis of the sample taken at the start (t=0) of the limit test showed a measured concentration of 0.83 mg/L, which was well above the water solubility. After 24 h of exposure the test concentration had decreased to 0.032 mg/L and further to below the limit of detection (Le. Below 0.03 mg/L) after 72 h of exposure. The average exposure concentration was calculated to be 0.068 mg/L, which was above water solubility.
-Mean cell densities: Table 3 shows mean cell densities measured at 24 h intervals at the different concentrations of the test substance.
-Inhibition of cell growth and reduction of growth rate: Table 4 shows the calculation of the percentages of inhibition of cell growth and Table 5 the percentages of growth rate reduction at different time intervals.
An average exposure concentration of 0.068 mg test substance per liter induced a statistically significant inhibition of cell growth of 19% (2 Sample t-Test, a = 0.05).19% inhibition is also considered to be biologically significant. An average exposure concentration of 0.068 mg test substance per liter induced a statistically significant reduction of growth rate of 4.2% (2 Sample t-Test, α = 0.05). However, 4.2% reduction is not considered to be biologically significant. Inhibition of cell growth and reduction of growth rate were (slightly) higher in the final test then in the range-finding test in a WAF prepared at a loading rate of 100 mg/L. Although initial concentrations were similar in both tests, the exact course of concentration decrease in time is not known. This might cause the difference in effect.
-Determination of effect concentrations: Table 6 shows the effect parameters based on both loading rate and average exposure
concentration.
-Experimental conditions: The pH was within the limits prescribed by the protocol (6.0-9.0, preferably not varying by more than 1.5 unit). The temperature of the test medium was 22.3°C at the start of the test. During the exposure period the temperature in the incubator was maintained between 23.6 and 23.8°C. Temperature remained within the limits prescribed by the protocol (21-25°C, constant within 2°C).

*Refer to "Any other information on results incl. tables" section for tables.

Results with reference substance (positive control):

Under the conditions of the reference study with Selenastrum capricornutum, potassium dichromate inhibited cell growth and reduced growth rate of this fresh water algae species at nominal concentrations of 0.18 and 0.56 mg/L, respectively, and higher. The EC50 for cell growth inhibition (EBC50: 0-72h) was 0.49 mg/L with a 95% confidence interval ranging from 0.28 to 0.85 mg/L. The historical ranges of the 72h EC50 for growth inhibition lie between 0.49 and 1.4 mg/L. Hence, the EBC50: 0-72h for the present batch corresponds with this range. The EC50 for growth rate reduction (ERC50: 0-72h) was 1.0 mg/L with a 95% confidence interval ranging from 0.58 to 1.8 mg/L. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/L. Hence, the ERC50: 0-72h for the present batch corresponds with this range.

Table 1. Mean cell densities (x10⁴cells/mL) during the range-finding test:

Nominal conc. of test substance (mg/L)	Exposure time (h)
	0	24	48	72
Blank-control	1	5	29.7	102.5
Solvent-control	1	4.9	31.4	111.3
0.1	1	4.9	30.8	113.4
1	1	5.1	30.8	107.3
10	1	4.5	24.1	157.2
100	1	4.8	25.1	139.7

Table 2.Percentage reduction of growth rate and inhibition of total growth during the range-finding test:

Nominal conc. of test substance (mg/L)	Cell growth (0-72 h)		Mean growth rate
	Mean area (A)	Inhibition (%)	µ (0-72 h)	Reduction (%)
Blank-control	2002.56		0.06416
Solvent-control	2148.80		0.06543
0.1	2159.08	-0.5	0.06570	-0.4
1	2088.36	2.8	0.06491	0.8
10	2514.24	-17	0.07012	-7.2
100	2333.92	-8.6	0.06851	-4.7

Table 3 .Mean cell densities (x 10⁴cells/mL) during the limit test:

Loading rate of the test substance (mg/L)	Exposure time (h)
	0	24	48	72
Blank-control	1	5.7	29.4	118.5
Solvent-control	1	6.2	30.9	136.5
100	1	6.3	23.8	110.8

Table 4. Percentage inhibition of cell growth during the limit test:

Loading rate of the test substance (mg/L)	Cell growth (0-72 h)
	Mean area (A)	Inhibition (%)
Blank-control	2204.38
Solvent-control	2468.34
100	1993.24	19.2

Table 5. Percentage reduction of growth rate at different time intervals during the limit test

Loading rate of the test substance (mg/L)	Mean growth rate
	µ (0-24 h)	Reduction (%)	µ (0-48 h)	Reduction (%)	µ (0-72 h)	Reduction (%)
Blank-control	0.07229		0.07038		0.06618
Solvent-control	0.07575		0.07137		0.06826
100	0.07680	-1.4	0.06599	7.5	0.06536	4.2

Table 6. Effect parameter

Parameter	Loading rate of the test substance (mg/L)	Average concentration of the test substance (mg/L)
NOEBC	-	-
72 h-EBC50	>100	>0.068
NOERC	100	0.068
72 h-ERC50	>100	>0.068

Validity criteria fulfilled:

yes

Remarks:

The cell density increased by an average factor of >16 within 3 d in controls. Further, test conditions (temperature and pH) remained within the ranges prescribed by the guideline

Conclusions:

Under the conditions of the study, the 72 h EC50 for both cell growth inhibition and growth rate 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 0.068 mg/L.

Executive summary:

A study was performed to assess the acute toxicity of the substance to the algae Selenastrum capricornutum according to the ISO International Standard 8692, OECD Guideline 201 and EU Method C.3, 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 exponentially growing algal cultures 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. Analysis of samples taken at the start of the test showed that the initial concentrations at nominal 10 mg/L and the WAF prepared at a loading rate of 100 mg/L were well above the water solubility of the test substance. These test concentrations did not remain stable during the 72-h test period. The results showed that both the EC50 for cell growth inhibition and growth rate reduction were expected to be above the water solubility of the test substance. 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, a limit test was performed using silanised glassware. In the limit test, exponentially growing algal cultures were exposed to a WAF prepared at a loading rate of 100 mg/L, a blank-control and a solvent-control. The initial algal density was 104 cells/mL and the total test period was 72 h. Analysis of the sample taken at the start of the limit test from the WAF showed a measured concentration of 0.83 mg/L. After 24 h of exposure the test concentration had decreased to 0.032 mg/L and further to below the limit of detection (i.e. below 0.03 mg/L) after 72 h of exposure. The average exposure concentration was calculated to be 0.068 mg/L, which was above water solubility. The test substance only induced 19% cell growth inhibition and 4% growth reduction following exposure to a WAF prepared at a loading rate of 100 mg/L, which corresponds to average exposure concentration of 0.068 mg/L. Therefore under the conditions of the study, the 72 h EC50 for both cell growth inhibition and growth rate 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 0.068 mg/L (Bouwman, 2004).

Description of key information

Based on the study conditions, the 72 h EC50 for both cell growth inhibition and growth rate 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 0.068 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.068 mg/L

Additional information

A study was performed to assess the acute toxicity of the substance to the algae Selenastrum capricornutum according to the ISO International Standard 8692, OECD Guideline 201 and EU Method C.3, 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 exponentially growing algal cultures 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. Analysis of samples taken at the start of the test showed that the initial concentrations at nominal 10 mg/L and the WAF prepared at a loading rate of 100 mg/L were well above the water solubility of the test substance. These test concentrations did not remain stable during the 72-h test period. The results showed that both the EC50 for cell growth inhibition and growth rate reduction were expected to be above the water solubility of the test substance. 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, a limit test was performed using silanised glassware. In the limit test, exponentially growing algal cultures were exposed to a WAF prepared at a loading rate of 100 mg/L, a blank-control and a solvent-control. The initial algal density was 104 cells/mL and the total test period was 72 h. Analysis of the sample taken at the start of the limit test from the WAF showed a measured concentration of 0.83 mg/L. After 24 h of exposure the test concentration had decreased to 0.032 mg/L and further to below the limit of detection (i.e. below 0.03 mg/L) after 72 h of exposure. The average exposure concentration was calculated to be 0.068 mg/L, which was above water solubility. The test substance only induced 19% cell growth inhibition and 4% growth reduction following exposure to a WAF prepared at a loading rate of 100 mg/L, which corresponds to average exposure concentration of 0.068 mg/L. Therefore under the conditions of the study, the 72 h EC50 for both cell growth inhibition and growth rate 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 0.068 mg/L (Bouwman, 2004).