Reaction products of n-octanol and acrylic acid, first distillation pitch

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 January 3, 2012 to February 5, 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study was performed according to the OECD 201 Guideline with GLP.

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 (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all concentrations.
- Sampling method: Samples for Total Organic Carbon (TOC) analysis were controlled at 0 h and 72 h, with a volume of 40 mL.
- Sample storage conditions before analysis: Samples were stored in a refrigerator until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
The test substance was not completely soluble in test medium at the loading rates initially prepared.
Preparation of test solutions started with loading rates ranging from 1.0 to 100 mg/L applying an overnight period of magnetic stirring to reach maximum solubility of the test substance in the test medium. The resulting aqueous mixtures were left to stabilize for 1½ to 2 hours were after the Water Accommodated Fractions (WAFs) were taken out by siphoning through glass wool. The lowest test solution for the combined limit/range-finding test was prepared by a 10-fold dilution of the WAF prepared at a loading rate of 1.0 mg/L. The final test solutions in the combined limit/range-finding test ranged from clear and colourless to slightly hazy. In the final test all solutions were clear and colourless besides the highest WAF which was slightly hazy.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture.

ACCLIMATION
- Acclimation period: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10E4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg CaCO3 per litre

Test temperature:

21.9 - 22.5 ºC

pH:

7.5 - 8.1

Nominal and measured concentrations:

WAFs prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass, containing 50 mL of test solution
- Initial cells density: 1 x 10E4 cells/mL.
- Control end cells density: The test solution in the highest WAF was slightly hazy which could disturb spectrophotometric measurement. Therefore algal density was determined by use of a microscope and a counting chamber throughout the test.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, M2.

TEST MEDIUM / WATER PARAMETERS
- Intervals of water quality measurement:
pH: At the beginning and at the end of the test. The pH of the solutions should preferably not deviate by more than 1.5 units during the test.
Temperature of medium: Continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Light intensity and quality: Continuously using TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 75 to 87 µE.m-2.s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Appearance of the cells: At the end of the final test microscopic observations were performed in the WAF prepared at 32 mg/L to observe for any abnormal appearance of the algae.


TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 10% of 1.0, 1.0, 10 and 100 mg/L.
- Results used to determine the conditions for the definitive study: The expected EL50 for growth rate reduction and yield inhibition was between WAFs prepared at loading rates of 10 and 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

ca. 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes

- Reduction of growth rate and inhibition of yield:
Growth rates were in the range of the controls at WAFs prepared at loading rates of 1.0 to 10 mg/L, whereas the growth rate of algae exposed to the higher WAFs were increasingly reduced. Statistically significant reduction of growth rate was found at the WAF prepared at 32 mg/L and higher (Bonferroni t-test, α = 0.05).
Inhibition of yield increased with loading rate of the test substance from 10 mg/L upwards resulting in 83% inhibition at the highest WAF. Statistically significant inhibition of yield was found at the loading rate of 10 mg/L and higher (Bonferroni t-test, α = 0.05).
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, The EC50 for growth rate reduction (ERC50: 0-72h) was 1.8 mg/L with a 95% confidence interval ranging from 1.4 to 2.4 mg/L. The EC50 for yield inhibition (EYC50: 0-72h) was 0.69 mg/L with a 95% confidence interval ranging from 0.50 to 0.95 mg/L.

Table 1. Mean cell densities (x 10⁴cells/ml) during the final test.

Test substance^1	Exposure time (hours)
Loading rate^2
(mg/l)	0	24	48	72
control	1.0	4.7	14.6	90.3
1.0	1.0	4.8	17.2	86.8
3.2	1.0	4.2	16.1	89.4
10	1.0	2.4	15.5	72.1
32	1.0	2.8	8.9	48.3
100	1.0	1.3	2.8	16.7

¹ Reaction products of n-octanol and acrylic acid, first distillation pitch.

² WAF prepared at the given loading rate.

Table 2. Percentage reduction of growth rate (total test period) and percentage inhibition of yield during the final test.

Test substance^1 Loading rate^2 (mg/l)	Mean growth rate		Yield (0-72 h)
	µ (0-72 h)	Reduction (%)	x10^4cells/ml	Inhibition (%)
control	0.06253		89.33
1.0	0.06196	0.9	85.83	3.9
3.2	0.06235	0.3	88.42	1.0
10	0.05941	5.0	71.08	20.4
32	0.05377	14.0	47.33	47.0
100	0.03843	38.5	15.67	82.5

¹ Reaction products of n-octanol and acrylic acid, first distillation pitch.

² WAF prepared at the given loading rate.

Table 3. Effect parameters.

Parameter	Test substance^1 Loading rate^2 (mg/l)
NOERL	10
72h-ERL10	Between 10 and 32
72h-ERL50	~ 100*
NOEYL	3.2
72h-EYL10	Between 3.2 and 10
72h-EYL50	Between 32 and 100

¹ Reaction products of n-octanol and acrylic acid, first distillation pitch.

² WAF prepared at the given loading rate.

* - approximated value based on the results of the combined limit/range-finding test and the final test.

Validity criteria fulfilled:

yes

Remarks:

(control, cell density increased by an average factor of >16 within 3 days; mean coefficient of variation for section-by-section specific growth rates (control) not exceed 35%.; coefficient of variation of average specific growth rates not exceed 7%)

Conclusions:

The EL50 for growth rate reduction (ERL50: 0-72h) approximated 100 mg/L.
The NOELR for growth rate reduction was 10 mg/L.

Executive summary:

Pseudokirchneriella subcapitata, Fresh Water Algal Growth Inhibition with test substance was carried out according to OECD 201 guideline and EU C.3 method (GLP study).

 

As the test substance was not completely soluble in test medium at the loading rates initially prepared, the method Water Accommodated Fractions (WAFs) was used. The concentration of test substance in the aquatic samples was calculated using the TOC content of pure test substance.

 

The test substance reduced growth rate and inhibited the yield of this fresh water algae species significantly at WAFs prepared at loading rates of 10 mg/L and higher.

 

The EL50 for growth rate reduction (ERL50: 0-72h) approximated 100 mg/L.

The EL50 for yield inhibition (EYL50: 0-72h) was between 10 and 32 mg/L.

The NOELR for growth rate reduction was 10 mg/L.

The NOELR for yield inhibition was 3.2 mg/L.

Description of key information

Key study: Experimental results: the OECD Guideline 201 and EU method C.3. GLP study. 
The EL50 for growth rate reduction (ERL50: 0-72h) approximated 100 mg/L. 
The NOELR for growth rate reduction was 10 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

10 mg/L

Additional information

Key study: Experimental results: the OECD Guideline 201 and EU method C.3. GLP study.

The EL50 for growth rate reduction (ERL50: 0-72h) approximated 100 mg/L.

The NOELR for growth rate reduction was 10 mg/L.