2,4-dimethyl-4-phenyltetrahydrofuran

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:

11 to 14 November 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Inspection: 31 July 2013/ Date of Signature: 20/01/14

Specific details on test material used for the study:

Storage: Refrigerated (2-8°C)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1.0, 3.2, 10, 32 and 100 mg/L
- Sampling method: At the start of the test (0 hours), 10 mL samples of freshly prepared test media were taken from the control and each test media preparation flask for chemical analysis.
At 72 hours, inoculated replicate test vessels for the control and each test concentration were pooled and samples (10 mL) taken for chemical analysis.
At each sampling occasion, duplicate samples were taken. One for chemical analysis and one as a ‘back up’ sample should further analysis be required.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: Pseudokirchneriella subcapitata (Strain 278/4)
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP)

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Test temperature:

21 to 24°C

pH:

7.96 to 10.46

Nominal and measured concentrations:

Nominal concentration: 1.0, 3.2, 10, 32 and 100 mg/L
Measured concentration (0 hr): 0.986, 3.00, 10.8, 30.9 and 93.0 mg/L
Measured concentration (72 hr): 1.13, 3.33, 11.6, 35.4 and 105 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 350 mL conical flasks
- Type (delete if not applicable): closed
- Initial cells density: 1 x 104 algae cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Light intensity and quality: 8200 lux at 0 hr and 7270 lux at 72 hr

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: particle counter (Z2 Coulter Counter®) and haemocytometer.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L.
- Results used to determine the conditions for the definitive study: Based on nominal concentrations, the results of the range-finding test suggested that the 72-hour ExC50 value would be between 1.0 and 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Zinc chloride

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

10 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.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Range-finding tests

The results of the range-finding test indicated that the 72-hour EC50 values for yield, specific growth rate and area under the curve (biomass) were likely to be between nominal concentrations of 1.0 and 100 mg/L.

Definitive Test

Chemical Analysis

The results of the chemical analysis are presented in the table below.

Table 6.1.5/1: Analysis of test substance in test media during the definitive test

Nominal concentration (mg/L)	Measured Concentration (mg/L)
	0 Hours (New media)	% of nominal concentration	72 Hours (Old media)	% of nominal concentration
Control	-	-	-	-
1.0	0.986	99	1.13	113
3.2	3.00	94	3.33	104
10	10.8	109	11.6	116
32	30.9	97	35.4	111
100	93.0	93	105	105

“- “: Test substance not detected above the limit of quantification (0.002 mg/L)

Analysis of the test samples at 0 and 72 hours showed measured concentrations to range from 93% to 116% of nominal. Given that the results were within the 80% to 120% of nominal acceptance criteria, and no decrease in measured concentration was observed over the 72-hour test period, the results have been based on nominal test concentrations only.

 

Environmental Conditions

The results of the environmental conditions and pH determinations are presented in table below.

Table 6.1.5/2: Environmental conditions and water quality during the definitive test with Pseudokirchneriella subcapitata

Nominal concentration (mg/L)	*Measured pH values
	0-hour		72-hour
Control	8.05	10.24	10.21	10.06
		10.01	9.92	10.08
1.0	8.01	10.46	9.66	9.92
3.2	8.03	10.45	10.35	10.37
10	8.09	9.65	9.74	9.96
32	8.12	8.03	8.08	8.04
100	8.14	7.96	8.03	7.98

* Measured pH values are determined using samples of test media from volumetric flasks at 0 hours and from each individual test vessel at 72 hours

All environmental conditions were within those specified in the study protocol. The OECD guideline states that the pH in the control should not vary by more than 1.5 units during the test. At the end of the test elevated pH values were observed in some of the vessels. This is an indicator of good algal cell growth, whereby the release of oxygen from the algal cells during normal photosynthetic activity promotes an alkali pH in the surrounding test media. It was considered that the elevated pH levels was due to good algal cell growth and therefore considered not to affect the validity of the test.

 

Test Media Descriptions

The freshly prepared test media at 0 hours appeared as colourless solutions. The appearance of the test media following the 72-hour exposure period are summarised in the table below.

Table 6.1.5/3: Test media descriptions at the end of the definitive test

Nominal concentration (mg/L)	Colour of test medium	State of test medium
	Inoculated with algal cells	Non-inoculated
Control 1.0 3.2 10 32 100	pale green	homogenous hazy dispersion of algae cells
	colourless	solution

 

Growth Test

Results of the growth test with Pseudokirchneriella subcapitata are presented in table below.

Table 6.1.5/4: Growth of Pseudokirchneriella subcapitata cultures during the definitive test

Nominal concentration (mg/L)	Cell concentration (cells/mL)			Mean cell concentration (cells x 10^4/mL)
	24-hour	48-hour	72-hour	24-hour	48-hour	72-hour
Control	28290	214900	454080	3.61	18.9	37.8
	39920	169320	360100
	41140	214760	381400
	27670	166240	309240
	47720	210200	439460
	31730	157280	321360
1.0	48300	48300	48300	3.88	19.7	48.4
	40910	40910	40910
	27080	27080	27080
1.0	44860	44860	44860	4.07	22.7	50.7
	41120	41120	41120
	36250	36250	36250
10	20890	20890	20890	2.28	9.52	31.6
	15220	15220	15220
	32370	32370	32370
32	16360	16360	16360	1.48	2.03	1.39
	13170	13170	13170
	14960	14960	14960
100	14150	14150	14150	1.29	1.21	1.53
	12370	12370	12370
	12020	12020	12020

 

Nominal concentration (mg/L)	Mean area under growth curve “A” (cells x 10^4/mL)			Average specific growth rate “μ” (cells x 10^-2/mL)
	0-24 hours	0-48 hours	0-72 hours	0-24 hours	0-48 hours	0-72 hours
Control	31.3	277	933	5.26	6.10	5.03
1.0	34.5	293	1090	5.53	6.19	5.39
3.2	36.9	335	1190	5.84	6.51	5.45
10	15.4	133	602	3.24	4.18	4.79
32	5.80	23.9	40.8	1.63	1.44	0.452
100	3.42	9.39	18.4	1.03	0.391	0.549

 

Table 6.1.5/5: Section-by-section growth rate of Pseudokirchneriella subcapitata in the control

Vessel Number	Section-by-section growth rates
	0-24 hours	24-48 hours	48-72 hours	0-72 hours
Control V1	0.043330	0.084486	0.031171	0.052996
Control V2	0.057679	0.060205	0.031441	0.049775
Control V3	0.058933	0.068856	0.023930	0.050573
Control V4	0.042407	0.074712	0.025862	0.047660
Control V5	0.065115	0.061780	0.030729	0.052541
Control V6	0.048112	0.066699	0.029772	0.048194
Mean	0.052596	0.069456	0.028818	0.050290
Standard deviation	0.009301	0.009011	0.003150	0.002193
Coefficient of variance (%)	17.68	12.97	10.93	4.36

 

Based on nominal test concentrations, the 72-hour E_yC₅₀and the 0-72-hour E_bC₅₀and E_rC₅₀values were calculated to be 14, 12 and 18 mg/L, respectively.

The corresponding NOEC values for yield, biomass and specific growth rate after 72 hours were 10, 3.2 and 10 mg/L, respectively.

 

Validity Criteria

The control cell density increased by a factor of 37. Therefore, the validity criterion of achieving an increase in control cell density by at least a factor of 16 was achieved, and the environmental conditions of the test were considered acceptable.

The mean coefficient of variation for section-by-section specific growth rates (0-24, 24-48 and 48-72 hours) in the control culture was 13.86% and therefore did not exceed 35%.

The coefficient of variation for average specific growth rate during the whole test period in replicate control cultures was 4.36% which did not exceed 7% and therefore the validity criteria were met.

Validity criteria fulfilled:

yes

Conclusions:

The effects of test substance on the growth of the unicellular green alga, Pseudokirchneriella subcapitata, were determined during a 72-hour growth inhibition toxicity test conducted in accordance with OECD Guideline No. 201 Alga, Growth Inhibition Test (adopted 23 March 2006) (Annex 5 corrected 28 July 2011).

All validity criteria were met therefore the test was considered valid. Based on nominal test concentrations, the 72-hour EyC50 and the 0-72-hour EbC50 and ErC50 values were calculated to be 14, 12 and 18 mg/L, respectively.

The corresponding NOEC values for yield, biomass and specific growth rate after 72 hours were 10, 3.2 and 10 mg/L, respectively.

A positive control test showed that the use of a modified test system to reduce losses of test substance through volatility (completely filled and sealed test vessels) had a slightly lowering effect on ECx values obtained compared with that in a conventional test system. As such it can be considered that the results obtained from the definitive test give a worst-case result for the test substance.

Executive summary:

The effects of test substance on the growth of the green alga, Pseudokirchneriella subcapitata, during a 72-hour growth inhibition test. The test was conducted in accordance with OECD Guideline No. 201. Alga, Growth Inhibition Test (adopted 23 March 2006) (Annex 5 corrected 28 July 2011).

 

The definitive test was conducted at nominal test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L. The concentrations used were based on the results of a range-finding test, for which only the key findings have been reported. A control group was also included.

 

Test vessels (350 mL conical flasks) were prepared which were completely filled and sealed to minimise any potential losses through volatility. Each test vessel was inoculated with 1 x 10⁴ algae cells/mL and incubated at 21 - 24°C (under 4440 – 8880 Lux light intensity) for 72 hours with cell counts at 24-hour intervals.

 

Analysis of the test media samples was conducted at 0 and 72 hours. Analysis of the test samples at 0 and 72 hours showed measured concentrations to range from 93% to 116% of nominal. Given that the results were within the 80% to 120% of nominal acceptance criteria, and no decrease in measured concentration was observed over the 72-hour test period, the results have been based on nominal test concentrations only.

 

The 72-hour yield (EyCx) and the 24, 48 and 72-hour biomass (EbCx) and growth rate (ErCx) toxicity values, with corresponding NOEC values, are presented in the table below.

Parameter	Toxicity values (mg/L)
	24-hour	48-hour	72-hour
Growth Rate
ErC10	4.3 (2.2 – 12)	4.6 (3.0 – 17)	10 (6.4 – 11)
ErC20	5.7 (3.2 – 18)	6.5 (2.4 – 19)	12 (11 – 13)
ErC50	14 (0.15 – 29)	16 (NC – 26)	18 (17 – 19)
NOEC	3.2	3.2	10

- Not applicable

(x - x) 95% confidence limits

NC Not calculated

 

Based on nominal test concentrations, the 72-hour E_rC₅₀ value was calculated to be 18 mg/L.

The corresponding NOEC value for specific growth rate after 72 hours was 10 mg/L.

 

A positive control test showed that the use of a modified test system to reduce losses of test substance through volatility (completely filled and sealed test vessels) had a slightly lowering effect on EC_x values obtained compared with that in a conventional test system. As such it can be considered that the results obtained from the definitive test give a worst-case result for the test substance.

 

All validity criteria were met therefore the test was considered valid.

Description of key information

OECD Guideline, 201, GLP, key study, validity 1:

ErC50(72h)=18 mg/L (based on nominal concentration confirmed analytically)

ErC10(72h)= 10 mg/L (based on nominal concentration confirmed analytically)

 

 

Key value for chemical safety assessment

EC50 for freshwater algae:

18 mg/L

EC10 or NOEC for freshwater algae:

10 mg/L

Additional information

To assess the toxicity of the registered substance to aquatic algae and cyanobacteria, one key experimental study is available.

The effects of test substance on the growth of the green alga, Pseudokirchneriella subcapitata, during a 72-hour growth inhibition test were assessed according to OECD Guideline No. 201 with GLP statement.

The definitive test was conducted at nominal test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L. The concentrations used were based on the results of a range-finding test, for which only the key findings have been reported. A control group was also included.

 

Analysis of the test media samples was conducted at 0 and 72 hours. Analysis of the test samples at 0 and 72 hours showed measured concentrations to range from 93% to 116% of nominal. Given that the results were within the 80% to 120% of nominal acceptance criteria, and no decrease in measured concentration was observed over the 72-hour test period, the results have been based on nominal test concentrations only.

 

Based on nominal test concentrations, the 72-hour E_rC₅₀ value was calculated to be 18 mg/L.

The corresponding NOEC value for specific growth rate after 72 hours was 10 mg/L.

 

All validity criteria were met therefore the test was considered valid.