4,4'-isopropylidenedi-o-cresol

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 Dec 2017

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:

28 Jul 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

National GLP Compliance Monitoring Authority, Govt. of India (22 Jul 2016)

Analytical monitoring:

yes

Remarks:

HPLC

Details on sampling:

- Concentrations: All test concentrations and the control.
- Sampling method: 10 mL were sampled from each replicate and mixed together for each group. The samples were centrifuged (2000 rpm for 10 min) to remove algae and divided into two portions, one of which was used for analysis and the other of which was stored.
- Sample storage conditions before analysis: -20 ± 5 °C

Vehicle:

yes

Remarks:

acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Four stock solutions (5.0, 10, 20, 40, and 80 mg/mL) were prepared by dissolving respective amounts of the test item in 1 mL/10 mL acetone. 12 µL from each stock were diluted to 120 mL with sterile algal culture medium to obtain the desired test concentrations. For the control, 1300 µL algal culture was diluted to 120 mL with sterile algal culture medium. For the vehicle control, 12 µL acetone and 1300 µL algal culture were diluted to 120 mL with sterile algal culture medium.
- Differential loading: Yes
- Controls: Algal culture without test item
- Chemical name of vehicle: Acetone
- Concentration of vehicle in test medium: 12 µL vehicle in 120 mL test medium
- Evidence of undissolved material: In the preliminary range finding test, precipitation was observed up to a concentration of 16.66 mg/L, while no precipitation was observed at the concentrations of 8.33 mg/L. Therefore, 8.33 mg/L was selected as the highest test concentration.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular freshwater green alga
- Strain: ATCC 22662
- Source: American Type Culture Collection, 10801, University of Boulevard, Manassas, Virginia, USA (10 Mar 2017)
- Age of inoculum (at test initiation): 2 d pre-culture, which was cultured under the same conditions as the test.
- Method of cultivation: Static condition. Pre-culture flasks were maintained at 21 - 23 °C and 6570 - 6627 lux with a universal UV-free white fluorescent lamp. The cells in the cultured flasks were maintained in suspension by agitating the test flasks continuously on an orbital shaker (100 ± 2 rpm).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21 - 23 °C

pH:

7.04 - 7.68

Nominal and measured concentrations:

Control, vehicle control, 0.50, 1.00, 2.00, 4.00, and 8.00 mg/L (nominal)
--- , --- , 0.57, 1.04, 2.21, 4.46, and 8.91 mg/L (measured after 72 h)

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL sterile conical flasks
- Initial cells density: 6554 cells/mL
- Control end cells density: 585417 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes, according to OECD guideline 201

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Culture medium was prepared by adding the required amounts of stock solutions I - IV and filling up to 1 L with sterilized deionised water, according to OECD guideline 201.
- Culture medium different from test medium: Culture medium same as test medium
- Intervals of water quality measurement: pH values were recorded at 0 and 72 h. Illumination and temperature were recorded daily using a light meter and a minimum - maximum thermometer.

OTHER TEST CONDITIONS
- Photoperiod: 24 h light/ d
- Light intensity and quality: 6550 - 6723 lux

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Visual cell count using a haemocytometer and a microscope after 0, 24, 48, and 72 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: Yes
- Test concentrations: 0.00 (control), 0.00 (vehicle control), 0.01, 0.1, 1.0, and 8.3 mg/L (2 replicates per test concentration)
- Results used to determine the conditions for the definitive study: Yes. The percent inhibition of biomass was 1.76, 2.16, 29.32 and 98.93 for the test concentrations of 0.01, 0.1, 1.0, and 8.33 mg/L, respectively.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.53 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.42 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control: Yes, the cell concentration in the control cultures and vehicle control cultures increased exponentially by a factor of 89.3 and 89.1.
- 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): 1.04 mg/L
- Other: The reference study dates from 10 - 15 Jun 2017

Reported statistics and error estimates:

The NOEC and LOEC were determined by a suitable statistical procedure for multi sample comparison. The following tests were applied to the biomass, specific growth rate and yield data: Bartlett's test for homogeneity of variance, Analysis of Variance (ANOVA) and Dunnett's Test. The EC values were calculated by Probit analysis.

VALIDITY CRITERIA

The study met the validity criteria laid down by the guideline (Table 1).

 

Table 1: Validity criteria for OECD 201.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	The cell concentration in the control cultures and vehicle control cultures increased exponentially by a factor of 89 .3 and 89 .1 within the test period.	Yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	The mean coefficients of variation for day 0-1, 1-2, and 2-3 in control culture and vehicle control cultures were 9.23 and 6.12%, respectively.	Yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus.	The coefficient of variation of average specific growth rates during the test period in the replicate control cultures and vehicle control cultures were 0.67 and 0.67%, respectively.	Yes

 

 

BIOLOGICAL RESULTS

Results are summarized in Table 2 and the obtained effect values for the toxicity to aquatic algae are summarized in Table 3.

 

Table 2. Percentage Inhibition of Biomass, Growth rate and Yield [%].

Nominal test concentration [mg/L]	Percentage [%] inhibition of
	Biomass EbC50 (72 h)	Growth rate ErC50 (72 h)	Yield EyC50 (72 h)
Control	-	-	-
Vehicle control	-	-	-
0.5	0.86	0.16	0.29
1.0	31.24	7.21	27.41
2.0	57.80	17.15	54.25
4.0	88.28	42.15	85.84
8.0	99.55	75.32	97.67

Note:Growth rate is a logarithmic term. Therefore, EbC and ErC values are not numerically comparable.

 

Table 3. Effect values for the toxicity to aquatic algae.

EbC10 (72 h)	0.81 mg/L
ErC10 (72 h)	1.53 mg/L
EyC10 (72 h)	0.92 mg/L
EbC50 (72 h)	1.75 mg/L
ErC50 (72 h)	4.42 mg/L
EyC50 (72 h)	2.04 mg/L
NOEC for biomass, growth rate and yield	0.5 mg/L
LOEC for biomass, growth rate and yield	1.0 mg/L

 

 

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Description of key information

OECD guideline, GLP compliant study. Pseudokirchneriella subcapitata were exposed to the test substance for 72 hours during the test. % of growth rate inhibition was recorded.

72h ECr50 value of 4.42 mg/L and 72h EC10 of 1.53 mg/L were calculated.

Key value for chemical safety assessment

EC50 for freshwater algae:

4.42 mg/L

EC10 or NOEC for freshwater algae:

1.53 mg/L