Allyl phenoxyacetate

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:

04 - 07 Jan 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2011

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatliches Gewerbeaufsichtsamt Hildesheim, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all concentrations at the start (0 h) and after 24, 48 and 72 h of exposure
- Sampling method for test item analysis: for the test item analysis at the beginning of the exposure and after 24 and 48 h separate replicates for each test item concentration and control were prepared with algae. For the test item analysis after 72 h, samples were taken from the test replicates.
- Sampling method for DOC analysis: the two highest concentration levels and the control were analytically verified via analysis of dissolved organic carbon (DOC, according to DIN EN 1484) at the start and at the end of the exposure. Separate replicates for the DOC measurement after 0 and 72 h were prepared without algae and incubated under test conditions.
- Sample storage conditions before analysis: all samples were stored at 6 ± 2 °C until the start of the analysis, if necessary. Prepared samples were stored at room temperature on the autosampler until analysis

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: stock solution of 100 mg/L was freshly prepared with dilution water and shaken until the test item was completely dissolved

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: HINDÄK, SAG 61.81
- Source (laboratory, culture collection): fresh stocks are prepared every month (culture originally from Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Germany)
- Age of inoculum (at test initiation): 4 d
- Method of cultivation: on Z-Agar, light intensity: 35-70 µE * m^-2 * s^-1 for 24 h/day

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21 - 22 °C

pH:

7.33 - 7.80 (in vessels with test item)
8.10 - 8.27 (in the control)

Nominal and measured concentrations:

0 (control), 12.0, 20.4, 34.6, 58.8 and 100 mg/L (nominal)
0 (control), 6.44, 12.5, 24.6, 44.1 and 76.9 mg/L (time weighted average mean measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL sterile Erlenmeyer flasks with cotton wool plugs
- Type (delete if not applicable): open
- Fill volume: 100 mL
- Initial cells density: 6153 cells/mL (nominal approx. 5 x 10^3 - 10^4 cells/mL)
- Control end cells density: 1849853 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Total organic carbon: 68.73% (calculated from the sum formula and the purity)
- Intervals of water quality measurement: pH was measured in one additional replicate of each test item concentration and the control at the start of the exposure while at the end it was measured from pooled samples of each test item concentration and the control after measurement of the cell density. Temperature was measured continuously. Light intensity was measured prior to the start of the exposure.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Photoperiod: 24 h day light
- Light intensity and quality: approx. 61.6 - 77.5 µE * m^-2 * s^-1, fluorescent tubes, Osram L 36 W-865, cool daylight
- Aggitation: oscillated on a rotary shaker at approx. 70 rpm

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination. Microscopic evaluation of the cells at the start and the end of exposure was carried out.
- Chlorophyll measurement: daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was found in the preliminary range finding test.

TEST CONCENTRATIONS
- Range finding study: yes, non GLP
- Test concentrations: 0.01, 0.1, 1, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: -1, 1, 0, -4 and 100% growth rate inhibition as well as -8, 4, -3, -25 and 100% yield inhibition at 0.01, 0.1, 1, 10, 100 mg/L, correspondingly. Based on these findings concentrations between 10 and 100 mg/L were used for the definitive study.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

24.9 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 24.5 - 29.0 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 6.44 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

12.7 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: <6.44-22.2 mg/L

Results with reference substance (positive control):

- Results with reference substance valid: yes
- ErC50: 0.564 mg/L (95% CI: 0.546 - 0.582 mg/L)
- EyC50: 0.322 mg/L (95% CI: 0.239 - 0.324 mg/L)

Reported statistics and error estimates:

All effect values are based on the time weighted average mean measured test item concentrations of the test item.
EC10-, EC20- and EC50-values with confidence intervals of growth rate and yield inhibition after 72 h were calculated by sigmoidal dose-response regression.
NOEC / LOEC was determined by calculation of statistical significance of biomass integrals, growth rate and yield. Following statistical tests were used: Hypothesis tests, monotonicity was done by trend analysis by contrasts (significance level 0.05); Shapiro-Wilk’s test on normal distribution was done with a significance level 0.01; Levene’s test on variance homogeneity was done with a significance level 0.01; William’s test on significant differences was done with a significance level 0.05; Welsh-t-test after Bonferroni-Holm was done with a significance level 0.05.

Table 1: Growth rate and yield inhibition at the different concentration levels after 72 h of exposure.

Time weighted average mean measured test item concentration [mg/L]	Replicate	Inhibition of growth rate [%]	Inhibition of yield [%]
76.9	1	100	100
	2	100	100
	3	100	100
	mean	n.a.	n.a.
44.1	1	100	100
	2	100	100
	3	100	100
	mean	n.a.	n.a.
24.6	1	53	95
	2	43	92
	3	47	93
	mean	48	94
12.5	1	11	47
	2	12	49
	3	12	51
	mean	12	49
6.44	1	8	38
	2	8	37
	3	9	39
	mean	8	38

The measured concentrations of the test item in the fresh media (0 h) were in the range of 97 to 101% of the nominal values at all tested concentration levels. The measured concentrations in the old media (72 h) were in the range of 12 to 61% of the nominal values of all concentrations. DOC measurements showed that the DOC content was in the range of 103 - 108% of the calculated TOC concentration at the start of the exposure and in the range of 102 - 110% of the calculated TOC concentration at the end of the exposure. It is therefore assumed that the test item might have degraded but was not lost via evaporation or adsorption.

Table 2: Measured concentrations of the test item over time and % of the nominal concentration.

Sampling date	0 h fresh medium		24 h old medium		48 h old medium		72 h old medium		Time weighted average mean measured concentrations [mg/L]
Nominal test item concentration [mg/L]	Meas. conc. [mg/L]	[%]	Meas. conc. [mg/L]	[%]	Meas. conc. [mg/L]	[%]	Meas. conc. [mg/L]	[%]
100	97.4	97	83	83	71.3	71	60.6	61	76.9
58.8	58.1	99	49.7	85	39.5	67	32.8	56	44.1
34.6	34.3	99	28.3	82	22.6	65	15.8	46	24.6
20.4	20.6	101	16.3	80	12.3	60	4.61	23	12.5
12	12.1	101	9.64	80	6.67	56	1.43	12	6.44
Control	< LOQ		< LOQ		< LOQ		< LOQ

* LOQ = 0.4 mg/L

Analytical results showed additional signals in the 24 h aged samples. One of these signals at 1.66 min increased after 48 and 72 h. It is assumed that this signal is a degradation product of the test item.

Table 3: DOC content of the test media at the beginning and at the end of the test.

Time weighted average mean measured test item concentration [mg/L]	Nominal calculated TOC concentration of the test item [mg C/L]	Test start (without algae), 0 h		Test end (without algae), 72 h
		DOC [mg C/L]	% of the nominal TOC concentration	DOC [mg C/L]	% of the nominal TOC concentration
76.9	68.7	70.5	103	69.3	102
44.1	40.4	43.8	108	44.5	110
Control	-	2.16		<2.00 (1.82)

* LOQ = 2 mg/L

Table 4: Further effect values and 95% confidence intervals of the test item.

Growth Rate Inhibition
	Time weighted average mean measured test item concentration [mg/L]
LOEC	=< 6.44
ErC20	20.6 (17.3-23.0)
Yield Inhibition
	Time weighted average mean measured test item concentration [mg/L]
NOEC	< 6.44
LOEC	=< 6.44
EyClO	< 6.44
EyC20	< 6.44
EyC50	12.8 (12.4-13.2)

Validity criteria fulfilled:

yes

Conclusions:

The test item was found to inhibit the growth rate of the green algae Pseudokirchneriella subcapitata after 72 h of exposure. The test (OECD 201, GLP) resulted in an ErC50 of 24.9 mg/L (meas. TWA) and an ErC10 of 12.7 mg/L (meas. TWA).

Executive summary:

The study was conducted according to OECD guideline 201, EU regulation No. 761/2009/Method C.3 and GLP. Pseudokirchneriella subcapitata was used as the test organism in a static test system. The green algae was exposed to nominal concentrations of 0 (control), 12.0, 20.4, 34.6, 58.8 and 100 mg/L (corresponding to 0 (control), 6.44, 12.5, 24.6, 44.1 and 76.9 mg/L TWA) for 72 h. An analytical monitoring via HPLC-DAD was performed in order to verify the concentrations in all test item concentrations and in the control after 0, 24, 48 and 72 h. Furthermore, DOC analysis was performed at the beginning and at the end of the exposure time for the two highest test concentrations and the control. HPLC-DAD results showed that the measured concentrations in the aged media (72 h) range between 12 and 61% of the nominal values. However, DOC analysis recovered 102 to 110% of the calculated TOC concentration at the end of the exposure. Therefore, it is assumed that the test item might have degraded but was not lost via evaporation or adsorption. Effect values were expressed in time weighted mean measured test item concentrations. Observed effects resulted in an ErC10 (72 h) of 12.7 mg/L and an ErC50 (72 h) of 24.9 mg/L.

Description of key information

ErC10 (72 h) = 12.7 mg/L (TWA, P. subcapitata, OECD 201)

ErC50 (72 h) = 24.9 mg/L (TWA, P. subcapitata, OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:

24.9 mg/L

EC10 or NOEC for freshwater algae:

12.7 mg/L

Additional information

A study is available, in which the toxicity of the substance to aquatic algae is controlled. The study was conducted according to OECD guideline 201, EU regulation No. 761/2009/Method C.3 and GLP. Pseudokirchneriella subcapitata was used as the test organism in a static test system. The green algae was exposed to nominal concentrations of 0 (control), 12.0, 20.4, 34.6, 58.8 and 100 mg/L (corresponding to 0 (control), 6.44, 12.5, 24.6, 44.1 and 76.9 mg/L TWA) for 72 h. An analytical monitoring via HPLC-DAD was performed in order to verify the concentrations in all test item concentrations and in the control after 0, 24, 48 and 72 h. Furthermore, DOC analysis was performed at the beginning and at the end of the exposure time for the two highest test concentrations and the control. HPLC-DAD results showed that the measured concentrations in the aged media (72 h) range between 12 and 61% of the nominal values. However, DOC analysis recovered 102 to 110% of the calculated TOC concentration at the end of the exposure. Therefore, it is assumed that the test item might have degraded but was not lost via evaporation or adsorption. Effect values were expressed in time weighted mean measured test item concentrations. Observed effects resulted in an ErC10 (72 h) of 12.7 mg/L and an ErC50 (72 h) of 24.9 mg/L.