COASOL 290 PLUS

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:

January 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: COASOL 290 PLUS
Chemical name: Reaction mass of dibutyl adipate and dibutyl glutarate
Batch/Lot Number: 6H04163A01
Description: Clear colourless liquid
Purity: 99.3 %
Expiry date: 04 August 2019
Storage condition: Room temperature (15-25 °C, ≤ 70 RH%)
Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to assure personnel health and safety.

Analytical monitoring:

yes

Details on sampling:

Samples were taken at all concentrations as well as from the control, at the beginning and at the end of the experiment.

Vehicle:

yes

Details on test solutions:

Test solutions were prepared using a saturated solution method (water accommodated fraction, WAF) according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23 (2000). Test item solutions (nominal loading rates of 1.0, 3.1, 9.8, 31.0 and 100.0 mg/L in the second run, used for the study conclusions) were prepared individually by dispersing/dissolving the amount of test item into the test medium (OECD Medium) two days before the start of the experiment. These solutions were shaken for about 24 hours at approximately 30°C and then equilibrated for about 24 hours at approximately 20°C. The non-dissolved test material was removed by filtration through a fine (0.22 µm) filter to give the appropriate WAF solutions. The test solutions were prepared and distributed into test vessels prior to introduction of algae.

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY. Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of Citoxlab Hungary Ltd.
Justification of species: The species of Pseudokirchneriella subcapitata used, being a fast-growing species, is convenient for culturing and testing and is a recommended species by relevant guidelines.
Culture conditions: Stock cultures are small algal colonies that are inoculated onto agar regularly. These are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines. The pre-culture is intended to give a quantity of algae suitable for the inoculation of test cultures. The pre-culture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, normally after an incubation period of about three days. When the algal cultures contain deformed or abnormal cells, they were discarded.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

Culture temperature was checked at the beginning of the experiment and each day thereafter in a flask filled with water, in the climatic chamber. In addition, water temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was between 22.2 and 22.4 °C measured in the flask and between 21.5 and 22.9 °C measured within the climate chamber.

pH:

The pH was checked at the beginning and at the end of the test, in the control and each concentration. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.37 –8.57 during the experiment.

Nominal and measured concentrations:

The following concentrations were tested in the second run: 1.0, 3.1, 9.8, 31.3 and 100.0 mg/L nominal loading rates WAFs. This range proved to be fully acceptable, covering biological results from no effects through to no cell growth.

Details on test conditions:

The test was performed with three replicates per test concentration and six replicates in the control group. Volumes of 100 mL algal suspension per replicate in 250 mL Erlenmeyer flasks were continuously shaken by a laboratory orbital shaker to keep algae in suspension. The flasks were covered with air-permeable stoppers. A concentration range-finding test was conducted to determine the approximate toxicity of the test item so that appropriate test concentrations could be selected for use in the definitive test. Algal cells were exposed to each concentration of the test item plus a control, for 72 hours. The test was performed with two replicates per each test concentration and three replicates in the control group.

The concentration levels used and results (72 h) of the preliminary range-finding test are summarised in the following table.

Nominal concentrations [mg/L nominal loading rate WAF] Untreated control 0.1 1 10 100
Average of cell number at 72 hours (x 104cell/mL) 72.00 71.00 69.50 68.50 3.50

Because significant inhibition was observed at the highest examined concentration level during the preliminary range-finding test, five test concentrations (6.25, 12.5, 25.0, 50.0 and 100.0 mg/L nominal loading rates WAFs) and one control were tested in the first main experiment. Due to greater toxicity than expected being observed during this experiment, the experiment was repeated using test item concentrations in a wider concentration range to fully cover the NOELR to the level with significant toxicity. Results of the first definitive test are not reported (but its full documentation will be stored as raw data under the present study code).

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

5.52 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

2.77 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

cell number

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

2.94 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

3.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

3.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Key result

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

3.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber. Microscopic observation of the algal cells in each concentration and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.

Validity criteria fulfilled:

yes

Executive summary:

The effect of COASOL 290 PLUS was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours. A significant toxic response was observed at the highest concentration level during the preliminary range-finding test, hence main study work was required. Two main experiments were run, each with five test concentrations and controls (6.25, 12.5, 25.0, 50.0 and 100.0 mg/L in the first, then 1.0, 3.1, 9.8, 31.3 and 100.0 mg/L in the second run, expressed as nominal loading rates WAFs). The test item was supplied as a reaction mass of dibutyl adipate and dibutyl glutarate. The test item was not fully soluble at all tested concentrations, hence a WAF method was used to produce a range of concentrations at nominal loading rates from 1.0 to 100 mg/L. As is typical for WAF studies with substances described as a ‘reaction mass’ it was not known in advance what fractions of the supplied substance may be soluble or may be toxic, hence a general analytical method was used to measure the total organic carbon per mL aquatic media (TOC). The biological results are reported in units of the nominal loading rates; the TOC results are considered to be supporting data, confirming exposure. Test concentrations as analytically determined by TOC analysis showed a linear relationship with the nominal concentrations in the range of biological relevance, giving full confidence that the solutions were prepared in a suitable manner. The test design included three replicates at each test concentration and six replicates for the untreated controls. Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (a= 0.05) by TOXSTAT software.TheE_rL_,E_bLand E_yL values of the test item and their confidence limits were calculated using Probit analysisby TOXSTAT software.

 

With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates, areas and yield were significantly different from that of the control group at the examined nominal loading rates of 3.1 – 100.0 mg/L, therefore the NOELR was determined as 1.0 mg/L nominal loading rate WAF and the LOELR was determined as 3.1 mg/L nominal loading rate WAF. The biological results are summarised in the following table:

Parameter (0-72 h)	Growth rate (r) [mg/L]		Yield (y) [mg/L]	Biomass (b) [mg/L]
		Calculation based on nominal loading rates WAFs
EL50	5.52		2.77	2.94
95 % conf. limits	4.85 – 6.28		2.47 – 3.10	2.58 – 3.34
NOELR	1.0		1.0	1.0
LOELR	3.1		3.1	3.1

Description of key information

The effect of COASOL 290 PLUS was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours. The test item was not fully soluble at all tested concentrations, hence a WAF method was used to produce a range of concentrations at nominal loading rates from 1.0 to 100 mg/L. With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates, areas and yield were significantly different from that of the control group at the examined nominal loading rates of 3.1 – 100.0 mg/L, therefore the NOELR was determined as 1.0 mg/L nominal loading rate WAF; the LOELR was determined as 3.1 mg/L nominal loading rate WAF. The 72-hour EL50 for growth was established to be 5.52 mg/L, for yield 2.77 mg/L and for biomass 2.94 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

5.52 mg/L

EC10 or NOEC for freshwater algae:

1 mg/L