isostearic acid monoisopropanolamide

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From June 11, 1999 to July 09, 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

HPLC analysis and UV/VIS-detection based on the main compound

Details on sampling:

Analytical report 726164 (Schreitmiiller, 1999)
Analvses of the test substance concentrations:
Duplicate samples from the freshly prepared test media of all test concentrations and from the control were taken at the start of the test (without algae). To confirm the maintenance of the test substance concentrations throughout the test duration, additional flasks with adequate volumes of the freshly prepared test media of all test concentrations and the control were incubated under the same conditions as in the actual test (but without algae). From these flasks, duplicate samples were taken at the end of the test (after the 72 hours test period). Since in a pre-experiment the concentration of the test substance had slightly decreased after storage at -20 “C, acetonitrile was added immediately after sampling to stabilize the test substance during storage. Then the samples were deep-frozen (at about -20 “C).
The concentrations of the test substance were measured in the duplicate test medium samples from all test concentrations from both sampling times (0 and 72 hours). From the control samples only one of the duplicate samples was analyzed from each of both sampling times (0 and 72 hours). Remaining samples are kept stored at about -20 “C to enable additional analyses.

Vehicle:

no

Details on test solutions:

An aliquot of the test substance was warmed up by means of a waterbath to about 40°C for 5 minutes to increase the fluidity and to improve the spreading properties. The test medium of the highest test concentration of nominal 100 mg/L was prepared by dispersing 70 mg test substance in 700 ml test water by ultrasonic treatment for 30 minutes and intense stirring for 30 minutes at room temperature. Adequate volumes of this test medium were diluted with test water to prepare the test media with the lower test substance concentrations of 32, 10, 3.2, 1.0, and 0.32 mg/L nominal. The test media were prepared just before inoculation of the algae (start of the test). Additionally, a control was tested in parallel (test water without addition of the test substance).
The test concentrations were based on the results of a range-finding test. The enlarged spacing factor of 3.2 between the test concentrations was chosen, since according to the results of the range-finding test a large concentration range had to be tested in the definitive test.

Test organisms (species):

other: Scenedesmus subspicatus CHODAT, Strain No. 86.81 SAG

Details on test organisms:

The test was started (0 hours) by inoculation of 10,000 algal cells per mL test medium. These cells were taken from an exponentially growing pre-culture, which was set up 3 days prior to the test at the same conditions as in the test.

Test type:

other: stirred by magnetic stirrers

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol/l (= 24 mg/L) as CaC03

Test temperature:

22°C

pH:

7.9-8.7

Nominal and measured concentrations:

0.32, 1.0, 3.2, 10, 32 and 100 mg/L (nominal)

Details on test conditions:

The test design included three replicates per test concentration and six replicates in the control. Volumes of 15 mL algal suspension per replicate were continuously stirred by magnetic stirrers in 50 mL Erlenmeyer flasks. The flasks were covered with glass dishes. They were incubated in a temperature controlled water bath at 22 “C, and continuously illuminated at a measured light intensity of about 8020 Lux (mean value), range: 7500 to 8530 Lux (minimum and maximum value of measurements at 9 places distributed over the experimental area at the surface of the test media). This illumination was achieved by fluorescent tubes (Philips TLD 36W/840), installed above the water bath in a distance of about 35 cm from the test flasks. The test duration was 72 hours.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 126 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 2.6 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: biomass and growth rate

Remarks on result:

other: 0.19 mg/L measured

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.42 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 0.31 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Analytical results:
The analytically determined mean test substance concentrations in the test media ranged at the start of the test from 69 to 97% of the nominal values and at the end of the test from 46 to 72%. The total mean measured test concentrations (calculated as the average over all measurements per test concentration) varied in the range of 58 to 81% of the nominal values. Therefore, the reported biological results are related to the total mean measured test substance concentrations. These were 0.19 mg/L (0.32 mg/L nominal), 0.68 mg/L (1.0 mg/L nominal), 2.6 mg/L (3.2 mg/L nominal), 7.5 mg/L (10 mg/L nominal), 23 mg/L (32 mg/L nominal), and 78 mg/L (100 mg/L nominal).
Biological results:
The test substance had a statistically significant inhibition effect on the growth (biomass and growth rate) of Scenedesmus subspicatus after the exposure period of 72 hours first at the concentration of 0.68 mg/L (72h LOEC). The 72h NOEC was 0.19 mg/L, since at this test concentration both the mean biomass and the mean growth rate u of the algae were statistically not significantly lower than in the control. The EC-values were calculated for both parameters the algal biomass b and the growth rate u after 72 hours test duration:
- EC50: 2.6 and 126 mg/L*, respectively
- EC10: 0.31 and 0.42 mg/L, respectively
(* this EC-value should be taken with caution, since the inhibition of ~1 was up to the highest test concentration lower than 50%)
At the microscopic examination of the shape of the algal cells after 72 hours test period, no difference was observed between the algae growing in the test concentration of nominal 1.0 mg/L and the algal cells in the control. Thus, the shape of the algal cells growing at least up to this test concentration was obviously not affected.
ln the control, the cell density has increased from nominal N = 1 x 10E04 cells/mL at the start of the test (0 hours) to N = 72 x 10E04 cells/mL (mean value) after 72 hours and therefore the factor of validity of 16 given by the guidelines was exceeded. Thus, the algal growth in the control was sufficiently high under the test conditions.
The test media of the test concentrations of nominal 0.32 to 3.2 mg/L were determined to be clear test media. At the concentration of nominal 10 mg/L, a slight turbidity was observed and at the concentration of nominal 32 and 100 mg/L, a distinct turbidity was present and the test substance was visually determined to be distributed homogeneously. However, the analytical results indicated possible non-homogeneities in the dispersion of the test substance in the test media.

None.

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the nominal 72 h ErC50 and NOEC were determined to be 126 and 0.32 mg/L (0.19 mg/L measured), respectively.

Executive summary:

A study was conducted to evaluate the toxicity of the test substance, isoC18 MIPA (94.1% active), to the green algae Scenedesmus subspicatus according to OECD Guideline 201 and EU Method C.3, in compliance with GLP. The algal cells were exposed to 0.32, 1.0, 3.2, 10, 32 and 100 mg/L (nominal) test substance for 72 h. The test design included three replicates per test concentration and six replicates in the control. Volumes of 15 mL algal suspension per replicate were continuously stirred by magnetic stirrers in 50 mL Erlenmeyer flasks. Test substance concentrations were verified analytically by HPLC with UV/VIS detection. Inhibition of algae growth was determined from the area under the growth curves (biomass) and the specific growth rates for exponentially growing cultures. The analytically determined mean test substance concentrations ranged from 69 - 97% of nominal values at test start to 46 - 72% of nominal values at test end. The total mean measured test concentrations (calculated as the average of all measurements per test concentration) varied in the range of 58 - 81% of nominal values. Therefore, the reported biological results were related to the total mean measured test substance concentrations. These were 0.19 mg/L (0.32 mg/L nominal), 0.68 mg/L (1.0 mg/L nominal), 2.6 mg/L (3.2 mg/L nominal), 7.5 mg/L (10 mg/L nominal), 23 mg/L (32 mg/L nominal) and 78 mg/L (100 mg/L nominal). The test substance had a statistically significant inhibition effect on the growth (biomass and growth rate) of the algae during the exposure period of 72 h at 1.0 mg/L and above. The growth of the algae in the controls met the validity criteria. Slight to distinct turbidity was observed in the test media at 32 mg/L and above. The estimated ErC50 should be taken with caution, since the inhibition of the growth rate was lower than 50% at this concentration. Under the study conditions, the nominal 72 h ErC50 and NOEC were determined to be 126 and 0.32 mg/L (0.19 mg/L measured), respectively (Batscher, 1999).

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

From April 06, 2002 to April 22, 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to the section 13 for details on the category justification.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 4.6, 10, 22, 46 and 100 (% WSF of 100 mg/L loading rate)
- Sampling method: During the definitive test, singular samples for TOC-analysis were taken from all test groups (incubated without algae) according to the schedule presented below:
- Sampling frequency: 0, 24 and 72 h
- Sampling volume: 40 mL
- Sample storage: Samples were stored in a refrigerator until analyses

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Preparation of test solutions started with an aqueous mixture at a loading rate of 100 mg/L. This mixture was magnetically stirred for 48 h and then left to settle overnight. This resulted in a hazy dispersion with a floating layer and precipitate. Subsequently, the hazy water phase was collected by siphoning it through glass wool. This resulted in a slightly hazy solution that was used for testing. The lower test concentrations were prepared by subsequent dilution of the Water Soluble Fraction (WSF). The final test solutions were all clear and colourless except for the undiluted WSF that was slightly hazy. 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 10(4) cells/mL.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: NIVA CHL 1
- Source: In-house laboratory culture:
- Method of cultivation: Algae stock cultures were started by inoculating growth medium (M1) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light (60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm) in a climate room at a temperature of 21-24°C.

ACCLIMATION
- Acclimation period: 3 d
- Culturing media and conditions (same as test or not): Yes (M2 media according to the OECD 201 was used)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

Test temperature:

21.8 and 23.9°C

pH:

7.4-8.1 (measured at the beginning and the end of the test)

Nominal and measured concentrations:

Measured concentration (Time weighted average): 0, 1, 1.7, 3.9 and 9.4 mg/L containing 0, 10, 22, 46 and 100% WSF (Water Soluble Fraction) , respectively. For 4.6% WSF, TWA concentration was not measured.
For details on measured concentrations versus nominal concentrations, please refer to 'table 1' in the 'Over all remarks' section

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL
- Type (delete if not applicable): Open
- Material, size, headspace, fill volume: 100 mL glass vessel containing 50 mL of test solution
- Initial cells density: 10(4) cells/mL
- No. of vessels per concentration (replicates): 3 replicates of each test concentration
- No. of vessels per control (replicates): 6 replicates of the control and 4 or 5 replicates of each test concentration without algae for sampling purposes (analytical monitoring).

GROWTH MEDIUM
- Standard medium used: Yes (M2)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water purified by reverse osmosis (Milli-RO)
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination
- Light intensity and quality: Using TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 63 to 73 E.m-2.s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a Varian Cary 50 single beam spectrophotometer with immersion probe (pathlength =20 mm). Algal medium was used as blank.
- Other: At the end of the final test microscopic observations were performed on the test concentration closest to the EC50 to verify a normal and healthy appearance of the inoculum culture and to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Aprrox. 2
- Range finding study: Yes, six replicates of exponentially growing algae were exposed to a control and a WSF (Water Soluble Fraction) prepared at 100 mg/L.
- Test concentrations: Three replicates/concentration were exposed to dilutions containing 0.1, 1.0 and 10% of the WSF
- Others: Test procedure and conditions were similar to those applied in the final test with the following exceptions except: (1). pH was only measured in the control and the highest test concentration. (2). At the end of the test algae were not observed to verify a normal and healthy appearance (3) Samples for possible analysis were only taken from the control and the highest test concentration.
- Results used to determine the conditions for the definitive study: The final test was performed based on the results of the combined limit/range-finding test. For details on the results, please refer to 'table 1' and 'table 2' under 'Any other information on material and method incl. tables'.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 9.4 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 2.3 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI: 1.3-4.0 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 3.4 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: CI: 2.1-5.3 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 1 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Details on results:

- Growth rate: Growth rates were in the range of the controls at the TWA concentrations up to and including 1.0 mg/L during the 72 h test period, whereas the growth rate of algae exposed to 1.7 mg/L and higher were increasingly reduced with a maximum reduction of 40.6% at a TWA concentration of 9.4 mg/L. Statistically significant reduction of growth rate was found at TWA test concentrations of 1.7 mg/L and higher.

- Yield: Inhibition of yield increased with increasing concentration of test material from 1.7 mg/L upwards resulting in 87 % inhibition at the highest test concentration of 9.4 mg/L.. Statistically significant inhibition of yield was found at TWA test concentrations of 1.7 mg/L and higher. Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.

Results with reference substance (positive control):

- Results with reference substance valid?: Yes
- EC50:
1. The EC50 for growth rate reduction (ErC50: 0-72h) was 1.2 mg/L with a 95% confidence interval ranging from 0.77 to 2.0 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.
2. The EC50 for yield inhibition (EyC50: 0-72h) was 0.44 mg/L with a 95% confidence interval ranging from 0.37 to 0.54 mg/L. The historical ranges of the 72h EC50 for yield inhibition lie between 0.43 and 1.1 mg/L. Hence, the EYC50: 0-72h for the present batch corresponds with this range.

Reported statistics and error estimates:

Following statistical methods were employed:
- Chi-Square test for normality
- Levene's test for homogeneity of variance
- Bonferroni t-Test

Table1:  Mean cell densities (x10⁴cells/mL) during the combined limit/range-finding test

Test group  (% WSF of 100 mg/L loading rate)	Exposure time (h)
	0	24	48	72
Control	1.0	5.5	27.1	161.5
0.10	1.0	5.3	24.2	147.4
1.0	1.0	5.0	21.7	148.9
10	1.0	5.1	22.7	145.4
100	1.0	3.0	9.9	32.0

Table 2: Percentage reduction of growth rate and inhibition of yield during the combined limit/range-finding test

Test group  (% WSF of 100 mg/Lloading rate)	Mean growth rate		Yield (0-72 h)
	µ (0-72 h)	Reduction (%)	x10^4cells/mL	Inhibition (%)
Control	0.07052		160.48
0.10	0.06931	1.7	146.35	8.8
1.0	0.06944	1.5	147.89	7.8
10	0.06914	2.0	144.44	10.0
100	0.04795	32.0	31.00	80.7

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

Test group (% WSF of 100 mg/L loading rate)	Test group TWA conc. (mg/l)	Exposure time (h)
		0	24	48	72
Control	-	1.0	9.3	39.1	143.4
4.6	n.m.	1.0	9.4	42.0	147.4
10	1.0	1.0	8.6	41.1	139.9
22	1.7	1.0	6.0	28.7	104.5
46	3.9	1.0	5.5	11.9	62.6
100	9.4	1.0	3.8	5.3	19.5

Table 4: Percentage reduction of growth rate (total test period) and percentage inhibition of yield during the final test

Test group (% WSF of 100 mg/L loading rate)	Test group TWA conc. (mg/L)	Mean growth rate		Yield (0-72 h)
		µ (0-72 h)	Reduction (%)	x10^4cells/mL	Inhibition (%)
Control	-	0.06896		142.40
4.6	n.m.	0.06932	-0.5	146.35	-2.8
10	1.0	0.06856	0.6	138.94	2.4
22	1.7	0.06450	6.5	103.55	27.3
46	3.9	0.05743	16.7	61.55	56.8
100	9.4	0.04098	40.6	18.46	87.0

Table 5: Percentage reduction of growth rate at different time intervals during the final test

Test group (% WSF of 100 mg/L loading rate)	Test group TWA conc. (mg/L)	Mean growth rate
		µ (0-24 h)	Reduction (%)	µ (24-48 h)	Reduction (%)	µ (48-72 h)	Reduction (%)
Control	-	0.09286		0.05993		0.05409
4.6	n.m.	0.09329	-0.5	0.06244	-4.2	0.05224	3.4
10	1.0	0.08934	3.8	0.06542	-9.2	0.05092	5.9
22	1.7	0.07428	20.0	0.06543	-9.2	0.05379	0.5
46	3.9	0.07084	23.7	0.03130	47.8	0.07015	-29.7
100	9.4	0.05387	42.0	0.01343	77.6	0.05563	-2.8

n.m. Not measured

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 72 h ErC50 for growth rate was > 9.4 mg/L, beyond the range tested and therefore above the maximum solubility of substance in test medium under the study conditions. The 72 h ErC10 was 2.3 mg/L (95% CI: 1.3 – 4.0 mg/L). The 72 h EbC50 was equivalent to 3.4 mg/L (95% CI: 2.1 - 5.3 mg/L) and the NOEC for both growth rate and yield was 1.0 mg/L.

Executive summary:

A study was conducted to determine the toxicity of the read across substance, C8-18 and C18-unsatd. MIPA, to freshwater green algae (Pseudokirchneriella subcapitata) according to OECD Guideline 201, in compliance with GLP. The substance was not completely soluble in test medium at the concentrations tested and hence the preparation included a 2 d magnetic stirring period followed by overnight settlement and subsequent collection of the Water Soluble Fraction (WSF). Exponentially growing algal cultures were exposed to 0, 4.6, 10, 22, 46 and 100% of a WSF prepared at a loading rate of 100 mg/L. The total test period was 72 h and the initial algal cell density was 10E+4 cells/mL. Samples for measurement of Total Organic Carbon (TOC) were taken at the start, then after 24 and 72 h of exposure. The time weighted average (TWA) exposure concentrations were calculated to correspond to 1.0, 1.7, 3.9 and 9.4 mg/L for the test groups containing 10, 22, 46 and 100% WSF, respectively. Growth rate and yield were significantly inhibited at a concentration of 1.7 mg/L (TWA) and higher. Under the study conditions, the 72 h ErC50 for growth rate was > 9.4 mg/L, beyond the range tested and therefore above the maximum solubility of substance in test medium under the study conditions. The 72 h ErC10 was 2.3 mg/L (95% CI: 1.3 – 4.0 mg/L). The 72 h EbC50 was equivalent to 3.4 mg/L (95% CI: 2.1 - 5.3 mg/L) and the NOEC for both growth rate and yield was 1.0 mg/L (Migchielsen, 2010). Based on the results of the read across study, the test substance is also expected to have similar toxicity to algae.

Key value for chemical safety assessment

EC50 for freshwater algae:

9.4 mg/L

EC10 or NOEC for freshwater algae:

2.3 mg/L

Additional information

A study was conducted to determine the toxicity of the read across substance, C8-18 and C18-unsatd. MIPA, to the freshwater green algae Pseudokirchneriella subcapitata according to OECD Guideline 201, in compliance with GLP. The substance was not completely soluble in test medium at the concentrations tested and hence the preparation included a 2 d magnetic stirring period followed by overnight settlement and subsequent collection of the Water Soluble Fraction (WSF). Exponentially growing algal cultures were exposed to 0, 4.6, 10, 22, 46 and 100% of a WSF prepared at a loading rate of 100 mg/L. The total test period was 72 h and the initial algal cell density was 10E+4 cells/mL. Samples for measurement of Total Organic Carbon (TOC) were taken at the start, then after 24 and 72 h of exposure. The time weighted average (TWA) exposure concentrations were calculated to correspond to 1.0, 1.7, 3.9 and 9.4 mg/L for the test groups containing 10, 22, 46 and 100% WSF, respectively. Growth rate and yield were significantly inhibited at a concentration of 1.7 mg/L (TWA) and higher. Under the study conditions, the 72 h ErC50 for growth rate was > 9.4 mg/L, beyond the range tested and therefore above the maximum solubility of substance in test medium under the study conditions. The 72 h ErC10 was 2.3 mg/L (95% CI: 1.3 – 4.0 mg/L). The 72 h EbC50 was equivalent to 3.4 mg/L (95% CI: 2.1 - 5.3 mg/L) and the NOEC for both growth rate and yield was 1.0 mg/L (Migchielsen, 2010).

A study was conducted to evaluate the toxicity of the test substance, isoC18 MIPA (94.1% active), to the green algae Scenedesmus subspicatus according to OECD Guideline 201 and EU Method C.3, in compliance with GLP. The algal cells were exposed to 0.32, 1.0, 3.2, 10, 32 and 100 mg/L (nominal) test substance for 72 h. The test design included three replicates per test concentration and six replicates in the control. Volumes of 15 mL algal suspension per replicate were continuously stirred by magnetic stirrers in 50 mL Erlenmeyer flasks. Test substance concentrations were verified analytically by HPLC with UV/VIS detection. Inhibition of algae growth was determined from the area under the growth curves (biomass) and the specific growth rates for exponentially growing cultures. The analytically determined mean test substance concentrations ranged from 69 - 97% of nominal values at test start to 46 - 72% of nominal values at test end. The total mean measured test concentrations (calculated as the average of all measurements per test concentration) varied in the range of 58 - 81% of nominal values. Therefore, the reported biological results were related to the total mean measured test substance concentrations. These were 0.19 mg/L (0.32 mg/L nominal), 0.68 mg/L (1.0 mg/L nominal), 2.6 mg/L (3.2 mg/L nominal), 7.5 mg/L (10 mg/L nominal), 23 mg/L (32 mg/L nominal) and 78 mg/L (100 mg/L nominal). The test substance had a statistically significant inhibition effect on the growth (biomass and growth rate) of the algae during the exposure period of 72 h at 1.0 mg/L and above. The growth of the algae in the controls met the validity criteria. Slight to distinct turbidity was observed in the test media at 32 mg/L and above. The estimated ErC50 should be taken with caution, since the inhibition of the growth rate was lower than 50% at this concentration.Under the study conditions, the nominal 72 h ErC50 and NOEC were determined to be 126 and 0.32 mg/L (0.19 mg/L measured), respectively(Batscher, 1999).