Octanoic acid, 2-phenoxyethyl ester

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:

20 Jan - 13 Nov 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Remarks:

Undissolved test material was visible and is expected to have caused a reduction of algal growth by physical effects. The determined effect values do not reflect the actual toxicity of the substance du to testing above the limit of water solublitiy.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth 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

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Water samples of 2 mL were taken at 0, 24 and 72 h (test 1) and at 0, 6, 24 and 72 h (test 2), respectively. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Sample storage conditions before analysis: Samples were stored in a freezer until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
Range finding test: Preparation of test solutions in the combined limit/range-finding test started with a stock solution prepared at a loading rate of 100 mg/L applying 48 hours of magnetic stirring followed by a stabilisation period of ~ 24 hours. The resulting solution was slightly hazy with a floating layer. The test solution was then siphoned off and lower test concentrations were prepared by subsequent dilutions of the saturated solution.

In the two final tests, preparation of test solutions started with a stock solution prepared at a loading rate of 100 mg/L applying 24 hours of magnetic stirring followed by a stabilisation period of ~ 24 hours. To minimise hydrolytic degradation the pH was set to pH 6.1 - 6.2 before the start of stirring. The resulting solution was slightly hazy and contained undissolved material. The test solution was siphoned off, pH was set to 6.3 - 6.5 and lower test concentrations were prepared by subsequent dilutions of the saturated solution i.

- Evidence of undissolved material:
(I) Range-finding test: The undiluted test solution was slightly hazy whereas the dilutions were clear and colourless.
(II) Final tests: The final test solutions were colourless to slightly hazy and generally contained undissolved material at concentrations containing 10% ofthe undiluted test solution and higher.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: freshwater 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 ware continuously aerated and exposed to light (60 – 120 µE/m2/s) in a climate room at a temperature of 21 - 24 °C.

ACCLIMATION
- Acclimation period: 4 days
- Culturing media and conditions: Cells from the algal stock culture ware inoculated in culture medium (M2 according to OECD 201) at a cell density of 10000 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/L as CaCO3

Test temperature:

Test 1: 22.5 - 23.1 °C
Test 2: 22.1 - 23.0 °C

pH:

Test 1: 6.5 - 6.6 (start) - 4.4 - 4.7 (end)
Test 2: 6.1 - 7.0 (start) - 4.5 - 4.7 (end)

Nominal and measured concentrations:

Nominal (test 1 and test 2): 4.6, 10, 22, 46 and 100% of a 100 mg/L loading rate saturated test solution
Measured (initial):
Test 1 after 0 h: 0.976, 0.765, 3.42, 3.19 and 16-18.9 mg/L
Test 1 after 24 h:
Test 2 after 0 h: 0.168, 0.438, 1.05, 2.38 and 5.02 - 5.88 mg/L
Test 2 after 6 h: Test 2 after 24 h:

Details on test conditions:

TEST SYSTEM
- Test vessel: not further specified
- Material: all-glass; Size: 100 mL; Fill volume: 50 mL
- Initial cells density: 1000 cells/mL
- Control end cells density: 69.3 x initial density
- No. of vessels per concentration: 3
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: yes/no
- Detailed composition if non-standard medium was used: M2 as recommended by OECD 201

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-Ro water (tap-water purified by reverse osmosis)
- Culture medium different from test medium: yes
- Intervals of water quality measurement: The pH was measured at the beginning and at the end of the test. Temperature was measured continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes
- Light intensity and quality: continuous light using TLD-lamps of the type "cool-white" of 30 Watt with a light intensity within the range of 88 - 90 µE/m2/s (test 1) and 70 - 84 µE/m2/s (test 2)

EFFECT PARAMETERS MEASURED: Algal growth were measured after 24, 48 and 74 h test duration.
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. As the test solutions in the 1st study were turbid and disturbed spectrophotometric measurement, algal density at 24, 48 and 72 hours of exposure was determined by use of a microscope and a counting chamber. In the 2 final study test solutions were less turbid and could therefore be measured applying spectrophotometric measurement.

TEST CONCENTRATIONS
- Range finding study: performed
- Test concentrations: 100 mg/L loading rate syphoned test solution and dilutions containing 0.1, 1.0 and 10% of the undiluted test solution
- Results used to determine the conditions for the definitive study: The actual measured concentration was below the limit of detection. The EC50 for growth rate reduction and yield inhibition was expected to exceed the concentration present in a syphoned test solution prepared at a loading rate of 100 mg/L and thus expected to be above the limit of solubility.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.4 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: WSF

Basis for effect:

growth rate

Remarks on result:

other: Test 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

10 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: WSF

Remarks:

based on the measured initial concentration in the undiluted WSF (19 mg/I).

Basis for effect:

growth rate

Remarks on result:

other: Test 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

54 other: % of WSF with a nominal loading rate of 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WSF with a nominal loading rate of 100 mg/L

Basis for effect:

growth rate

Remarks on result:

other: Test 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

22 other: % of WSF with a nominal loading rate of 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WSF with a nominal loading rate of 100 mg/L

Basis for effect:

growth rate

Remarks on result:

other: Test 1

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.
- Effect concentrations exceeding solubility of substance in test medium: test 1: Undissolved material was observed in the form of flakes in the undiluted test solution.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 1.8 mg/L (1.4 - 2.5 mg/L)
-other: The historical range for growth rate reduction is between 0.82 and 2.3 mg/L. Hence, the 72 h-EC50 for the present batch corresponds with this range.

The pH of the control and all test concentrations exceeded the lower limit at the end of the test. However, the algal growth was not inhibited in the control group (linear growth curve).

Test 1: During the test algal lumps were visible in all test groups after 48 hours and also in the control group after 72 hours. In addition, undissolved material was observed in the form of flakes in the undiluted test solution. Based on both the visual observations and the analytical results it appeared obvious that effects on algal growth in the final experiment were related to testing above solubility rather than to toxic effects of the substance tested.

Test 2: The concentrations measured during the second study confirmed the results obtained in the first study. Despite the fact that vessels ware pre-incubated with test solutions, the concentrations declined within six hours of exposure below the LOD (<0.007 mg/l). Hence, degradation of the test substance rather than adsorption was probably the most likely reason for the decreasing concentrations. Accordingly, the results of test 1 was used for the determination of NOEC and EC50 value.

The conducted tests showed that the effects an algal growth were likely to be caused by physical rather than toxic effects and due to undissolved material. Hence, the EC50 for both growth rate reduction and yield inhibition exceeded the limit of solubility.

Table 1: Mean cell densities

Percentage of 100 mg/L loading rate syphoned test solution [%]	Exposure time (h)
	0 h	24 h	48 h	72 h
Test 1
control	1.0	4.6	28.5	69.3*
4.6	1.0	4.8	25.7*	56.9*
10	1.0	5.4	30.8*	59.6*
22	1.0	6.8	31.0*	58.1*
46	1.0	5.4	24.3*	26.8*
100	1.0	4.6	1.4*	1.2*
Test 2
Control	1.0	4.8	26.5	96.7
4.6	1.0	5.2	27.9	39.6*
10	1.0	4.8	24.4	48.5*
22	1.0	4.8	23.1	55.1*
46	1.0	5.2	25.0	41.9*
100	1.0	5.9	28.0	40.3*

* Small lumps of clotted algae were observed.

Table 2: Concentrations of test substance in test medium

Time of sampling (h)	Percentage of 100 mg/L loading rate siphoned test solution [%]	Measured concentration [mg/L]**
Test 1
0	0	< LOD
	4.6	0.976
	10	0.765
	22	3.42
	46	3.19
	100	18.9
	100*	16.0
Test 2
0	0	< LOD
	4.6	0.168
	10	0.438
	22	1.05
	46	2.38
	100	5.02
	100*	5.88

*: without algae

**: All test substance concentrations measured on days 24, 48 and 72 were under the level of detection.

Conclusions:

The conclusion of the final test was that the EC50 for both growth rate reduction and yield inhibition exceeded the limit of solubility. Effects observed in part of the solutions were related to testing above solubility rather than to intrinsic toxic effects of the test substance.
An additional full study confirmed the above results: effects on algal growth were most likely related to physical rather than toxic effects. Hence, the EC50 for both growth rate reduction and yield inhibition exceeded the limit of solubility.

Description of key information

No toxic effects on aquatic algae up to the limit of water solubility. Physical effects were observed.

Additional information

The acute toxicity of 2-Phenoxyethyl octanoate (CAS No. 23511-73-1) to the algae Pseudokirchneriella subcapitata was tested in a GLP guideline study following OECD 201. The study report includes two full tests hereinafter referred to as test 1 and test 2. In test 2 the test vessels were pre-incubated with test solutions to prevent a possible adsorption of test substance. The stock solutions for both tests were prepared by adding 100 mg test substance/L. The water soluble fraction (WSF) was siphoned off and the test substance concentrations were prepared by diluting the WSF. The prepared nominal test substance concentrations contained 4.6, 10, 22, 46 and 100% of the stock solution with a loading rate of 100 mg/L. The test concentrations were slightly hazy and concentrations >10% WSF contained undissolved test material.

The test concentrations were analytically verified by HPLC. In test 1 the measured test concentrations after 0 h were: 0.976, 0.765, 3.42, 3.19 and 16-18.9 mg/L whereas after 24 hours the concentrations were below the detection limit (LOD: 0.06 mg/L). The decrease in test concentration was related to a possible adsorption of the test substance to the test vessels. Therefore the test vessels were pre-incubated with test solutions in test 2 to prevent a possible adsorption of test substance. In addition, a sampling occasion was included after 6 hours of exposure to enable more accurate assessment of the expected decline in test concentrations. The measured test concentrations in test 2 were 0.168, 0.438, 1.05, 2.38 and 5.02 - 5.88 mg/L at test start but were also below the detection limit after 6 hours. In both tests algal lumps were visible after 48 hours at all tested concentrations and also in the control group after 72 hours. The algal growth rates in the test concentrations containing 46% and 100% WSF were reduced compared to the control. Based on this observation an EC50 value and a NOEC related to the algal growth rate in test 1 were determined. The reported EC50(72 h) was 54% of WSF. An EC50(72 h) of 10 mg/L was recalculated based on the initially measured concentration of 19 mg/L in the undiluted WSF (assumption: 100% WSF= 19 mg/L ; 54% WSF = 19 mg/L x 54% = 10 mg/L). The NOEC(72 h) was 22% of WSF (NOEC (72 h): 3.4 mg/L based on initial measured concentration).

 

According to the reported conclusion the EC50 for both growth rate reduction and

yield inhibition exceeded the limit of solubility of the test substance. Based on both the visual observations and the analytical results it appeared obvious that effects on algal growth in the final experiment were related to testing above solubility rather than to toxic effects of the substance tested. The observed effects are most likely cause by physical rather than toxic effects.