Glycerides, C8-18 and C18-unsatd., reaction products with ethylene oxide and glycerol

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:

weight of evidence

Study period:

From February 6, 2012 to March 23, 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

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: On the day of analysis, the samples were defrosted at room temperature. The test samples were diluted in a 1:3 (v:v) ratio with acetonitrile and analysed. If necessary, the samples were further diluted with 75/25 (v/v) acetonitrile/M2-medium to obtain concentrations within the calibration range.
- Sample storage conditions before analysis: The samples were stored in the freezer (≤ -15°C)

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The test substance was not completely soluble in test medium at the initial loading rates prepared.
Preparation of test solutions started with individually prepared loading rates between 1.0 and 100 mg/L that were magnetically stirred for a period of 22-23 hours. The resulting aqueous mixtures were then left to settle for 2 hours. The resulting Water Accommodated Fractions (WAFs) were then collected by means of siphoning the middle fractions. The final test solutions were in general slightly hazy for the WAFs prepared at 22 mg/L and higher and appeared clear and colourless at the WAFs prepared at 10 mg/L or lower. Note that on two consecutive days test solutions were prepared for the final test. The first set was used to pre-incubate the test vessels. After one day of pre-incubation, the solutions were discarded and replaced with freshly prepared solutions as to minimise possible adsorption of the test substance to glass.
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 10E4 cells/mL.

Test organisms (species):

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

Details on test organisms:

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

ACCLIMATION
- Acclimation period: 4 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 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.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

Test temperature:

21.4 - 22.8 ºC

pH:

6.2 - 8.1

Nominal and measured concentrations:

WAFs prepared at loading rates of 4.6, 10, 22, 46 and 100 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL
- Material, size, headspace, fill volume: all-glass, containing 50 mL of test solution.
- Initial cells density: 1 x 10E4 cells/mL
- 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 63 to 69 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 on all test concentrations to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 1.0, 10 and 100 mg/mL
- Results used to determine the conditions for the definitive study: The expected EC50 for growth rate reduction approximated the concentration in a WAF prepared at 100 mg/L and the expected EC50 for yield inhibition was between WAFs prepared at 10 and 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 43 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 2.3 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Growth rate: Growth rates were in the range of the controls at WAFs prepared between 4.6 and 22 mg/L during the 72-hour test period, whereas the growth rate of algae exposed to WAFs prepared at 46 and 100 mg/L were increasingly reduced. Statistically significant reduction of growth rate was found at WAFs prepared at 46 mg/L and higher.

Inhibition of yield: No inhibition of yield was observed up to and including WAFs prepared at 22 mg/L. Significant and increasing inhibition of yield was observed at WAFs prepared at 46 and 100 mg/L. Statistically significant inhibition of yield was found at WAFs prepared at 46 mg/L and higher.

- Other: 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:
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.

Loading rate WAF* (mg/l)	Exposure time (hours)
	0	24	48	72
Control	1.0	3.7	10.2	30.6
4.6	1.0	3.3	11.5	39.3
10	1.0	3.7	10.2	41.1
22	1.0	3.6	9.7	35.7
46	1.0	3.7	11.2	19.7
100	1.0	3.3	8.0	15.3

* WAF prepared at the given Glycerides, coco mono- and di-, ethoxylated loading rate.

 

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

 

Loading rate WAF* (mg/l)	Mean growth rate		Yield (0-72 h)
	µ (0-72 h)	Reduction (%)	x10^4cells/ml	Inhibition (%)
Control	0.04745		29.63
4.6	0.05091	-7.3	38.33	-29.4
10	0.05150	-8.5	40.08	-35.3
22	0.04963	-4.6	34.67	-17.0
46	0.04129	13.0^#	18.67	37.0^#
100	0.03738	21.2^#	14.25	51.9^#

* WAF prepared at the given Glycerides, coco mono- and di-, ethoxylated loading rate.

^#Statistically significant from control (α=0.05).

 

Table 3. Effect parameters.

 

Parameter	Loading rate WAF* (mg/l)	Average measured conc. (mg/l)	Measured concentration at 0 h (mg/l)
NOERC	22	n.d.	n.d.
72h-ERC10	> 22, < 46	n.d.	n.d.
72h-ERC50	> 100	> 2.3	> 43
NOEYC	22	n.d.	n.d.
72h-EYC10	> 22, < 46	n.d.	n.d.
72h-EYC50	> 46, < 100	~ 2.3	~43

* WAF prepared at the given Glycerides, coco mono- and di-, ethoxylated loading rate.

n.d. Not determined.

 

Validity criteria fulfilled:

yes

Remarks:

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

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, Glycerides, coco mono- and di-, ethoxylated reduced growth rate and inhibited the yield of this fresh water algae species significantly at WAFs prepared at a loading rates of 46 mg/l and higher.

The EC50 for growth rate reduction (ERC50: 0-72h) exceeded a WAF prepared at a loading rate of
100 mg/l, corresponding with an average measured exposure concentration of 2.3 mg/l and an initial (study start) concentration of 43 mg/l.

The EC50 for yield inhibition (EYC50: 0-72h) approximated a WAF prepared at a loading rate of
100 mg/l, corresponding with an average measured exposure concentration of 2.3 mg/l and an initial (study start) concentration of 43 mg/l.

The NOEC for both growth rate reduction and yield inhibition equalled the concentration present in a WAF prepared at a loading rate of 22 mg/l. A corresponding average measured exposure concentration could not be determined.

The use of the average measured exposure concentration in the calculation of the EC50 value had a significant effect on the outcome of the study. Care should however be taken in the interpretation of the results of the study based on average measured test concentrations as, whilst there was a significant decline in measured test concentrations over the test period, it was not possible to determine the exact cause for this decline. Based on this decline, the following result based on the 0 h measured test concentration is proposed: EC50 value for growth rate reduction > 43 mg/l.

Executive summary:

The Pseudokirchneriella subcapitata, Fresh Water Algal Growth Inhibition with Glycerides, coco mono- and di-, ethoxylated was determined according to the OECD Guideline 201, with GLP. The test substance was not completely soluble in test medium at the initial loading rates prepared. Preparation of test solutions started with individually prepared loading rates between 1.0 and 100 mg/L. A final test was performed based on the results of a preceding combined limit/range-finding test. Six replicates of exponentially growing algal cultures were exposed to a control, while three replicates per test group were exposed to WAFs prepared at loading rates of 4.6, 10, 22, 46 and 100 mg/L. The total test period was 72 hours and the initial algal cell density was 10E04cells/mL. Samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Under the conditions of the present study with Pseudokirchneriella subcapitata, Glycerides, coco mono- and di-, ethoxylated reduced growth rate and inhibited the yield of this fresh water algae species significantly at WAFs prepared at a loading rates of 46 mg/l and higher.

The EC50 for growth rate reduction (ERC50: 0-72h) exceeded a WAF prepared at a loading rate of

100 mg/l, corresponding with an average measured exposure concentration of 2.3 mg/l and an initial (study start) concentration of 43 mg/l.

The EC50 for yield inhibition (EYC50: 0-72h) approximated a WAF prepared at a loading rate of

100 mg/l, corresponding with an average measured exposure concentration of 2.3 mg/l and an initial (study start) concentration of 43 mg/l.

The NOEC for both growth rate reduction and yield inhibition equalled the concentration present in a WAF prepared at a loading rate of 22 mg/l. A corresponding average measured exposure concentration could not be determined.

The use of the average measured exposure concentration in the calculation of the EC50 value had a significant effect on the outcome of the study. Care should however be taken in the interpretation of the results of the study based on average measured test concentrations as, whilst there was a significant decline in measured test concentrations over the test period, it was not possible to determine the exact cause for this decline. Based on this decline, the following result based on the 0 h measured test concentration is proposed: EC50 value for growth rate reduction > 43 mg/l.

Description of key information

Weight of evidence: Experimental results: OECD Guideline 201 and EU method C.3. GLP studies.
The EC50 for growth rate reduction (ERC50: 0-72h) is between 10-100 mg/L.

Additional information

Weight of evidence: Experimental results: OECD Guideline 201 and EU method C.3. GLP studies.

The EC50 for growth rate reduction (ERC50: 0-72h) is between 10-100 mg/L.