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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 November 2010 and 29 March 2011
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to official EC and OECD test guidelines, and in compliance with GLP.

Data source

Reference Type:
study report

Materials and methods

Test guidelineopen allclose all
according to guideline
EU Method C.3 (Algal Inhibition test)
according to guideline
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Diisoamyl phthalate
Diisoamyl phthalate
Details on test material:
- Name of test material (as cited in study report): Diisoamyl phthalate
- Substance type: Organic
- Physical state: Colourless to yellow liquid
- Analytical purity: 100%
- Batch No.: 7920-1-1
- Expiration date of the batch: 12 August 2012
- Storage condition of test material: Room temperature in the dark.

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Concentrations: Range finding test 1: 0.1, 1.0 and 10 mg/L
- COncentrations: Range finging test 2: 0.01, 0.1, and 1.0 mg/L.
- Concentrations: For definitive test: nominal concentration 1 mg/L and control
- Sampling method:

Test solutions


Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Common name: Unicellular green alga
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Axenic, uni-cellular, liquid slope cultures of algae obtained from laboratory in Scotland
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: The primary liquid cultures (100 ml) were incubated for approximately three days in an orbital incubator under continuous illumination at nominal temperatures in the range 21 to 25 ºC. This provided an inoculum in the log phase of growth, characterised by the cell density.

- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): Sterile algal nutrient medium.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
72 h

Test conditions

Test temperature:
The temperature in the media ranged from 21.1 to 23.3 ºC
The pH in the media ranged from 7.75 to 9.80.
Nominal and measured concentrations:
Range finding test:

Nominal concentration Measured concentrations
mg/L mg/L

0.01 0 hrs: 0.0323 72 hrs: nd(a)

0.1 0 hrs: 0.129 72 hrs: 0.0205

1.0 0 hrs: 0.636 72 hrs: 0.0789

Definitive test measured concentrations:

Nominal concentration Measured concentrations
mg/L mg/L

1.0 0 hrs: 0.220 72 hrs: nd(a)

1.0 0 hrs: 0.177 72 hrs: nd (a,b)

1.0 A 0 hrs: not applicable 72 hrs: 0.508, 0.534(b), 0.999, 0.179(b)

a: a value of half the limit of detection (0.025 mg/L) used in calculation of test results.
b: results of reserve samples due to unexpected results achieved from initial samples.
A: culture medium incubated under test conditions without algal cells.
Details on test conditions:
The test vessels (completely filled and sealed Wheaton vials of approximately 65 mL capacity) each containing control or test culture were placed in an illuminated orbital incubator according to a random number sequence. The cultures were incubated, without renewal of medium for 72 hours under continuous illumination (nominally 4440 to 8880 lux.) provided by 6 x 30 W “cool white” 1 metre fluorescent tubes. The temperature was maintained at 21 to 24°C.

Temperature and pH of control and test flasks at the start and end of the test were recorded. Suspension of the algal cells were ensured by the action of the orbital shaker, oscillating at a nominal 130 cycles per minute. The minimum and maximum temperature and light intensity (four corner positions and in a central position of the random block design) within the test area were determined each day. To minimise the impact of differences in light intensity across the test area on algal growth, control and test flasks were re-positioned in the test area each day during the definitive test.

Results and discussion

Effect concentrationsopen allclose all
72 h
Dose descriptor:
Effect conc.:
> 0.224 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
72 h
Dose descriptor:
Effect conc.:
ca. 0.224 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Details on results:
At the start of the test, the measured concentrations of diisoamyl phthalate in samples of the test cultures were 0.0323, 0.129 and 0.636 mg/L respectively; after 72 hours, the measured concentrations had decreased to between 8 and 16% of their starting values. At 1.0 mg/L, the mean measured level of diisoamyl phthalate was 0.224 mg/L.
The test results have been expressed in terms of mean measured concentration of diisoamyl phthalate. The calculated area under the growth curve, average specific growth rate and yield values are given and are expressed in terms of percentage inhibition by comparing the test group value with that of the control curve.

Applicant's summary and conclusion

Validity criteria fulfilled:
Diisoamyl phthalate was not found to be acutely toxic to P. Subcapitata at measured concentrations up to 0.224 mg/L, which were determined to be within its limit of solubility.
Consequently, after 72 hours of exposure the EbC50, ErC50 and EyC50 were > 0.224 mg/L.

The “no observed effect concentration” (NOEC) for area under the growth curve, growth rate and yield was 0.224 mg/L.

Executive summary:

The effect of diisoamyl phthalate (1,2-Benzenedicarboxylic acid, bis(3-methylbutyl) ester) on the growth of the unicellular green algae Pseudokirchnerirlla subcapitata was assessed under sealed conditions due to the volatile nature of the test substance.


The study was conducted in accordance with EC Methods for Determination of Ecotoxicity, Annex IV to Commission Regulation (EC) No 761/2009 (O.J. No. L220/36, 2009) Part C, Method 3 “Freshwater Algae and Cyanobacteria, Growth Inhibition Test” and Procedure 201 of the “Guidelines for Testing of Chemicals” of the Organisation for Economic Co-operation and Development: Freshwater Alga and Cyanobacteria, Growth Inhibition Test” (2006). The design of the study intentionally deviated from these guidelines in that the composition of the OECD medium and its pH were altered to suit the exposure regime, as outlined in OECD Guidance Document Number 23, Aquatic Toxicity Testing of Difficult Substances and Mixtures ENV/JM/MONO(2000)6.


Diisoamyl phthalate was not found to be acutely toxic to P. Subcapitata at measured concentrations up to 0.224 mg/L, which were determined to be within its limit of solubility