2-methyl-4-phenyl-1,3-dioxolane

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:

25 Apr - 28 Jul 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatliches Gewerbeaufsichtsamt, Hildesheim, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Part 1: control, 6.25, 12.5, 25.0, 50.0, and 100 mg/L; Part 2: control, 1.563, 3.125, 6.25, 12.5, and 25.0 mg/L
- Sampling method: The test item analysis at the beginning of the exposure was done from additional replicates with algae from all test concentrations and the control. For the test item analysis after 72 h pooled samples from all replicates with algae from all test item concentrations and the control were used.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until sample preparation and at room temperature until the start of the analysis (in an autosampler), if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution of 100 mg/L was freshly prepared with dilution water and shaken manually until the solution was visually clear. For preparation the density of the test item was taken into account. Application was carried out by adding appropriate volumes of the stock solution to the replicate test vessels.
- Differential loading: no
- Controls: yes, dilution water without test item
- Evidence of undissolved material: no

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: unicellular green alga
- Strain: SAG 61.81
- Source: Sammlung von Algenkulturen (SAG), Göttingen, Germany
- Method of cultivation: fresh stocks are prepared every month on Z-agar. Light intensity amounted to 2567 - 5130 lux for 24 h per day.
- Culture medium: nutrient medium Z according to Lüttge et al. (1994)
- Age of inoculum (at test initiation): a three days old preculture, prepared in dilution water, was used as inoculum.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol Ca+Mg/L

Test temperature:

Part 1: Mean = 21.8 °C, Min. = 21.5 °C, Max. = 22.0 °C
Part 2: Mean = 21.8 °C, Min. = 21.5 °C, Max. = 22.0 °C

pH:

Part 1: at 0 h: 8.00 (control), 7.86 - 8.09 (test concentrations), at 72 h: 8.18 (control), 7.65 - 8.15 (test concentrations)
Part 2: at 0 h: 7.93 (control), 7.90 - 7.92 (test concentrations), at 72 h: 9.05 (control), 8.62 - 8.95 (test concentrations)

Nominal and measured concentrations:

nominal Part 1: control, 6.25, 12.5, 25.0, 50.0, and 100 mg/L
nominal Part 2: control, 1.563, 3.125, 6.25, 12,5, and 25.0 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: sterile Erlenmeyer flask
- Type: sealed with cotton wool plugs
- Size, headspace, fill volume: Volume = 250 mL; Test volume = 100 mL; Headspace = 150 mL
- Initial cell density: Nominal: approximately 5E+03 - 5E+04 cells/mL; Current: 6305 cells/mL
- Control end cell density: Part 1: 2486523 cells/mL, Part 2: 2331925 cells/mL, after 72 h
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, nutrient medium Z according to Lüttge et al. (1994)

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: no
- Intervals of water quality measurement: At the start of the exposure, pH values were measured in one additional replicate of each test item concentration and the control. At the end of exposure, pH values were measured from pooled samples of each test item concentration and the control. The room temperature was measured continuously. Light intensity was measured prior to the start of exposure.

OTHER TEST CONDITIONS
- Photoperiod: 24 h light
- Light source: fluorscent tubes, OSRAM L 36 W/865, cool daylight
- Light intensity: approximately 4440 to 8880 lux, corresponding to 60 - 120 µE*m-2*s-1

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Fluorometer (Microplate Reader Chameleon V, Hidex, with Software Micro Win 4.41, Mikrotec Laborsysteme GmbH, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No auto-fluorescence was found in the preliminary range finding test at the highest concentration of 100 mg/L.
- Chlorophyll a-fluorescence: measured at 0, 24, 48, and 72 h
- Microscopic evaluation: at the start (0 h) and the end (72 h) of the incubation period.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: Yes, one non-GLP range finding test
- Test concentrations range finding test: 1.0, 10.0, and 100 mg test item/L (nominal)
- Results used to determine the conditions for the definitive study: yes, 100% growth rate inhibition and yield inhibition, respectively, after 72 h

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 50 - < 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

69.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI 95%

Remarks:

68.9 - 69.6 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.125 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

12.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

55.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% CI

Remarks:

44.7 - 69.1 mg/L

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities: no
- Any observations that might cause a difference between measured and nominal values: The tested concentration levels were all visually clear throughout the exposure.
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- ErC50 (72 h): 0.460 mg/L, 95% confidence interval 0.443 - 0.481 mg/L
- EyC50 (72 h): 0.317 mg/L, confidence interval 0.208 - 0.347 mg/L

Reported statistics and error estimates:

EC50 values and confidence intervals of growth rate inhibition after 72 h were calculated by straight line regression and for yield inhibition after 72 h by sigmoidal dose-response regression.
The NOEC and LOEC were determined by calculation of statistically significant differences of growth rates. As a standard, One Way Analysis of Variance (ANOVA) and Dunnett´s test were used for NOEC/LOEC calculations. When running an ANOVA, a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance tests were 0.05 and the alpha-value was 0.05.

In both parts the measured test item concentrations were within ± 20% of the nominal concentration. This indicates that the test item concentration was successfully maintained for the duration of the test.

Table 1: Evaluation of Part 1 after 72 h.Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

Nominal test item concentration [mg/L]	Replicate n°	Growth rate [d^-1]	Inhibition of growth rate [%]	Yield [cells/mL]	Inhibition of yield [%]
100	1	-0.088	100	-1466	100
	2	-0.056	100	-975	100
	3	-0.034	100	-609	100
	Mean	(+)  -0.059	100	(+) -1017	100
50	1	1.88	6	1762388	29
	2	1.90	5	1848368	25
	3	1.86	7	1661693	33
	Mean	(+)* 1.88	6	(+) 1757483	29
25.0	1	1.94	3	2097003	15
	2	1.96	1	2279280	8
	3	1.93	3	2025514	18
	Mean	(-)    1.94	2	(-) 2133932	14
12.5	1	1.85	7	1593643	36
	2	1.93	3	2081048	16
	3	1.94	3	2119330	15
	Mean	(-)    1.91	4	(+) 1931340	22
6.25	1	1.91	4	1960169	21
	2	1.86	7	1659438	33
	3	1.91	4	1958703	21
	Mean	(+)* 1.90	5	(+) 1859437	25
Control	1	1.97		2347387
	2	2.00		2510382
	3	2.02		2677492
	4	1.95		2162122
	5	2.01		2635320
	6	2.00		2548607
	Mean	1.99		2480218

* = biologically not significant, due to an inhibition of < 10%

 

Table 2: Measured Exposure Concentrations during the Definitive Test in fresh medium (0 h) and old medium (72 h), Part 1.

Sampling date	2016-04-25 0 h fresh medium		2016-04-28 72 h old medium
Start of analysis	2016-04-25		2016-04-28
Nominal concentration of the test item [mg/L]	Test substance
	meas. conc. [mg/L]	[%]	meas. conc. [mg/L]	[%]
100	103	103	97.7	98
50.0	53.9	108	50.4	101
25.0	27.1	109	25.4	102
12.5	13.6	109	13.3	106
6.25	6.64	106	6.41	103
Control	< LOQ		< LOQ

meas. conc.      = Measured concentration of the test item (dilution factor taken into account), mean value of two injections

%                        = Percentage of the nominal concentration of the test item

LOQ                    = Limit of quantification (2 mg test item /L)

Table 3:Evaluation of Part 2 after 72 h.Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

Nominal test item concentration [mg/L]	Replicate n°	Growth rate [d^-1]	Inhibition of growth rate [%]	Yield [cells/mL]	Inhibition of yield [%]
25.0	1	1.87	5	1704914	27
	2	1.91	3	1935790	17
	3	1.88	4	1777419	24
	Mean	(+)   1.89	4	(+) 1806041	22
12.5	1	1.91	3	1906698	18
	2	1.91	3	1937144	17
	3	1.91	3	1960767	16
	Mean	(+)   1.91	3	(+) 1934870	17
6.25	1	1.95	1	2161761	7
	2	1.96	1	2251180	3
	3	1.96	0	2253210	3
	Mean	(-)    1.96	1	(-) 2222050	4
3.125	1	1.97	0	2334002	0
	2	1.97	0	2325658	0
	3	1.96	0	2270180	2
	Mean	(-)   1.97	0	(-) 2309947	1
1.563	1	1.97	0	2299554	1
	2	1.96	0	2254732	3
	3	1.95	1	2206640	5
	Mean	(-)    1.96	0	(-) 2253642	3
Control	1	1.97		2305530
	2	1.93		2068509
	3	2.01		2600905
	4	1.96		2277566
	5	1.99		2436501
	6	1.96		2264711
	Mean	1.97		2325620

 

Table 4: Table 2: Measured Exposure Concentrations during the Definitive Test in fresh medium (0 h) and old medium (72 h) with algae, Part 2.

Sampling date	2016-07-25 0 h fresh medium		2016-07-28 72 h old medium
Start of analysis	2016-04-25		2016-04-28
Nominal concentration of the test item [mg/L]	Test substance
	meas. conc. [mg/L]	[%]	meas. conc. [mg/L]	[%]
25.0	28.2	113	25.3	101
12.5	13.6	109	12.9	104
6.25	6.80	109	6.51	104
3.125	3.45	110	3.16	101
1.563	1.60	102	1.59	102
Control	< LOQ		< LOQ

meas. conc.      = Measured concentration of the test item (dilution factor taken into account), mean value of two injections

%                        = Percentage of the nominal concentration of the test item

LOQ                    = Limit of quantification (2 mg test item /L)

 

Validity criteria fulfilled:

yes

Conclusions:

The study according to OECD 201 resulted in en ErC50 (72 h) of 69.2 mg/L and a NOErC (72 h) of 50 mg/L (nominal) based on inhibition of growth of Pseudokirchneriella subcapitata. The ErC10 (72 h) was not calculated but derived to be between 50 and 100 mg/L.

Description of key information

ErC50 (72 h) = 69.2 mg/L (nominal, OECD 201, Pseudokirchneriella subcapitata)

NOErC (72 h) = 50 mg/L (nominal, OECD 201, Pseudokirchneriella subcapitata)

Key value for chemical safety assessment

EC50 for freshwater algae:

69.2 mg/L

EC10 or NOEC for freshwater algae:

50 mg/L

Additional information

There is one GLP guideline study available, which determined the effects of the substance on the growth of unicellular freshwater green algae according to the principles of the OECD guideline 201. In a static test consisting of two parts, each with five concentrations in a geometrical series with a dilution factor of 2, Pseudokirchneriella subcapitata was exposed to the test substance for 72 h. Part 1 covered nominal concentrations ranging from 6.25 - 100 mg/L and part 2 covered the nominal concentrations ranging from 1.563 - 25.0 mg/L. Part 2 was carried out for the determination of the EyCx because in the first part of the study the calculation of the EyCx was not possible based on the results. The concentrations of the test item were analytically verified by HPLC-DAD analysis at the start (0 h) and end (72 h) of exposure. In both parts the measured test item concentrations were within ± 20% of the nominal concentration. This indicates that the test item concentration was successfully maintained for the duration of the test. Thus, the effect concentrations were based on the nominal values. The calculated nominal ErC50 (72 h) was 69.2 mg/L and the corresponding NOErC (72 h) was 50.0 mg/L, based on the inhibition of growth. The ErC10 (72 h) was between 50 - 100 mg/L.