2,2',6,6'-tetra-tert-butyl-4,4'-methylenediphenol

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 1987 to February 1988

Reliability:

2 (reliable with restrictions)

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: Miller, W. E. and Green, J . C. (1978) . The Selenastrum capricornutum (Prinz) algal bottle test. EPA-600/ 9-78-018 .

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES of the substance are all available in section 4 "Physical and chemical properties".

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Quantities of the test substance dissoved into Analar acetone
- Controls: + Analar acetone (0.1 ml/L max.)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Analar acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): adjusted to 0.1 ml/L.

Test organisms (species):

Scenedesmus capricornutum

Details on test organisms:

TEST ORGANISM
- Common name: Selenastrum capricornutum (Prinz)
- Strain: ATCC 22662
- Source (laboratory, culture collection): American Type Culture Collection, Maryland, Ohio, U.S.A. - Axenic stock culture
- Age of inoculum (at test initiation): In exponential phase growth measured using a Coulter Counter (Model TAII)
- Method of cultivation:
* Axenic stock culture: agar plates maintained under illumination (18h light, 6 h dark) at 18-22°C in a Gallenkamp vertical incubator. The cultures are renewed monthly.
*Cultures in liquid medium: initiated with cells from the agra plates. Then, grown as 50 mL batch cultures in 250 mL Erlenmeyer flasks for up to 3 days. The cultures are maintained at 22-26°C in a Gallenkamp orbital incubator under full constant illumination (3000 lux).

Test type:

other: agar plates

Water media type:

freshwater

Limit test:

no

Total exposure duration:

4 d

Hardness:

No indication in the study report

Test temperature:

22-27.6°C

pH:

7.6-7.9

Dissolved oxygen:

No indication in the study report

Salinity:

No indication in the study report

Nominal and measured concentrations:

- Test concentrations: 1, 2.2, 4.6, 10, 22, 46, 100, 220, 460, 1000 mg/L (nominal concentrations)

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks 250 mL of volume
- Material, size, headspace, fill volume: 50 mL of culture medium
- Incubation: Incubated in a cooled, orbital incubator (100 cycles/min)
- Initial cells density: 500 cells/mL
- No. of vessels per concentration (replicates): No replicates - 10 doses
- No. of vessels per control (replicates): 6 replicates


GROWTH MEDIUM
- Standard medium used: yes - Miller and Green (1978) (USEPA methods)

TEST MEDIUM / WATER PARAMETERS
- Preparation of the nutrient medium: By dissolving Analar grade salts in glass distilled deionised water. Nutrient concentrations were those described
by Miller and Green (1978) (USEPA methods) except that boric acid was at 105 mg/L instead of 184 mg/L in Miller & Green) and and sodium bicarbonate at
50 mg/L (instead of 15 mg/L in Miller & Green). The medium was sterile.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: constant illumination
- Light intensity and quality: 3000 lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The algal biomass was estimated by determination of the concentration of chlorophyll a following the method of Talling and driver, 1963
Talling, JF and Driver, D 1963 Some problems in the estimation of Chlorophyll a in phytoplankton. Proceedings, Conference of Primary Productivity Measurement Marine and Freshwater. Hawaii. 1961. USAEC TID (1963), 7633, pp 142-146.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Logarithmically spaced series
- Range finding study: no range finding study
- Test concentrations: 1, 2.2, 4.6, 10, 22, 46, 100, 220, 460, 1000 mg/L

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Chlorophyll a concentration

Results with reference substance (positive control):

No positive control

Concentration of Testing substance (mg/L)	Chlorophyll a conc. Day 4 (µg/L)	Chlrophyll a conc. on Day 4 as % of mean control value
Control	186	-
Control	179	-
Control	191	-
Control	56	-
Control	122	-
Control	228	-
1	193	120.4
2.2	195	121.5
4.6	155	96.6
10	203	126.9
22	196	122.6
46	202	125.9
100	215	134.5
220	209	130.2
460	175	109.6
1000	174	108.5

Validity criteria fulfilled:

yes

Executive summary:

The acute toxicity of 4,4'-methylene-bis-2,6-di-tert-butyl phenol to the planktonic alga, Selenastrum capricornutum was determined in a 4 day growth test. The 96h EC50 value based on Chlorophyll a concentrations on day 4 , was greater than 1000 mg/L the highest concentration tested.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Followed acceptable guideline and GLP requirements.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Qualifier:

according to guideline

Guideline:

other: ASTM Standard E1218-04

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Controls: Solvent control (dimethlyformamide in freshwater media; negative control freshwater AAP media)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): HPLC grade dimethylformamide
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): Primary stock was 50 mL DMF containing 32.6 ug test article per ml. Solvent control was freshwater AAP media containing 0.1 ml DMF per liter.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Not observed. Primary stock solution had a purple tint

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Freshwater green alga, Pseudokirchneriella subcapitata
- Source (laboratory, culture collection): Originally from University of Toronto Culture collection, and maintained at Wildlife International
- Age of inoculum (at test initiation): Cells taken from culture that had been transferred to fresh media 3 days prior to testing
- Method of cultivation: Maintained in freshwater AAP media. Growth had been monitored for at least 2 weeks prior to test and met standard criteria for healthy algal cultures

ACCLIMATION
- Acclimation period: at least 2 weeks prior to test
- Culturing media and conditions (same as test or not): same
- Any deformed or abnormal cells observed: not prior to test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

Well water used for the test media was moderately hard freshwater

Test temperature:

24 +/- 2 degrees C

pH:

pH of media was adjusted 7.5 +/- 0.1 with 10% hydrochlorid acid prior to use. pH ranged from 7.1 to 7.4 at test initiation, and from 8.3 to 8.5 at termination. The pH increase was typical for tests with this alga and was attributed to photosynthetic activity during the test.

Nominal and measured concentrations:

Nominal Mean Measured
Negative control < LOQ
Solvent Control 1.99 ng/L 1.50 ng/L
3.99 ng/L 3.35 ng/L
7.98 ng/L 6.84 ng/L
16.0 ng/L 12.9 ng/L
31.9 ng/L 26.5 ng/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml Erlenmeyer flasks with foam stoppers
- Material, size, headspace, fill volume: Glass, containing 100 ml test solution
- Initial cells density: 10,000 cells/ml
- Control end cells density: at 96 hours, negative control cell density was 3,806,667 cells/mL, and solvent control was 4,083,333 cells per mL. After three days, the mean cell density in the negative controls increased by a factor of 189 and after four days had increased by a factor of 382. This was greater than a factor of 16 and factor of 100 respectively required for a valid test.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes Freshwater AAP medium
- Detailed composition if non-standard medium was used:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Moderately hard well water from a well on-site. Water was passed
through a sand filter and pumped into 37,800 L storage tank. Prior to use, water
was filtered to 0.45 um to remove fine particles and passed through an ultraviolet sterilizer.
- Particulate matter: Filtered to 0.45 um
- Metals: met criteria
- Pesticides: met criteria
- Culture medium different from test medium: No
- Intervals of water quality measurement: temperature continuously in chamber, and twice daily in water. pH at beginning and end of test. Pesticides, metals, other water elements periodical assessment of well water.

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Adjustment of pH:
- Photoperiod: continuous lighting
- Light intensity and quality: Fluorescent lighint (cool white light tubes) at an intensity of 6000 lux +/- 10%


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: Hemacytometer and microscope or electronic particle counter
- Chlorophyll measurement:
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 5 test concentrations at a geometric progression
- Justification for using less concentrations than requested by guideline:
- Range finding study Yes
- Range finding Test concentrations: Negative control, 0.319, 3.19 and 31.9 ng a.i./L nominal
- Results used to determine the conditions for the definitive study: Highest concentration produced less than 1 % inhibition versus control; concentrations higher than the highest preliminary concentration would exceed water solubility

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 26.5 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: cell density

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 26.5 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

48 d

Dose descriptor:

EC50

Effect conc.:

> 26.5 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: cell density

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 26.5 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 26.5 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: cell density

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 26.5 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 26.5 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 26.5 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: cell density

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 26.5 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 26.5 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 26.5 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: cell density

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 26.5 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 26.5 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

ca. 26.5 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: cell density

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

ca. 26.5 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 26.5 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none noted
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no

Reported statistics and error estimates:

Test endpoints analyzed statistically were cell density, growth, growth rate and yield. Negative control and solvent control data for each parameter were compared with appropriate statistical tests. No significant differences were seen between the control groups (Student’s t-test, a = 0.05); therefore the control groups data were combined.
The 72 and 96 hour cell density, yield, and growth rate data were evaluated for normality and homogeneity of variances (a = 0.01) using Shapiro-Wilk’s and Levene’s tests respectively. Statistically significant differences between the control and treatment groups were identified using a test to compare treatment mean responses to the control response (Dunnett’s one-tailed t-test, a = 0.05). There were no statistical differences in reductions of cell density, yield or growth in the 96 hour test groups when compared with control. At 72 hours, Dunnett's test indicated there were significant reductions in cell density, yield, and growth rate in the 6.8 ng/L treatment group. Since this reduction was not dose-responsive, and appeared analagous to the data collected from other treatment groups, it was not considered treatment related.
Results of the statistical analysis were used to aid in determine the NOEC for each endpoint at 72 and 96 hours. Scientific judgment was also used to determine if statistical differences were biologically meaningful, and if the data showed a concentration-dependent relationship.

Results: After 72 hours of exposure, inhibition of cell density in the 1.50, 3.35, 6.84, 12.9 and 26.5 ng/L groups was 15, 8, 49, -9, and -3% respectively, relative to negative control. Inhibition of yield in the same gropus was 15, 8, 49 and -3% relative to negative control. Inhibition of growth rate in those groups was 4, 1, 13, -2 and -1% respectively relative to negative control.

After 96 hours, inhibition of cell density in the 1.50, 3.35, 6.84, 12.9, and 26.5 treatment groups was -23, -22, 12, -10, and 5% respectively, relative to the negative control. Inhibition of yield in the same groups was -24, -22, 12, -10, and -5%, respectively, relative to negative control. Inhibition of growth rate for the same test groups was -3, -4, 2, -2, and -1% respectively, relative control.

As statistical analysis showed no significant reduction in cell density, yield or growth rate at 96 hours in the treatment groups compared to control, the 96 hour NOEC values for cell density, yield and growth rate was 26.5 ng/L. At 72 hours, there was a statistically significant reduction in cell density, yield, and growth rate only for the 6.8 ng/L test group. Since the reduction was not dose-responsive, and appeared analagous to data from the other treatment groups, it was not considred treatment related, and the 72 hour NOEC for cell density, yield and growth rate was also determined to be 26.5 ng/L.

Resulting EC50 and NOEC calculations based on mean measured concentrations of test article

	Cell Density		Growth Rate		Yield
Exposure Duration (hours)	EC50 (ng/L)^1	95% Confidence Interval (ng/L)^1	EC50 (ng/L)^1	95% Confidence Interval (ng/L)^1	EC50 (ng/L)^1	95% Confidence Interval (ng/L)^1
24	> 26.5	n/a^3	> 26.5	n/a^3	--^2	--^2
48	> 26.5	n/a^3	> 26.5	n/a^3	--^2	--^2
72	> 26.5	n/a^3	> 26.5	n/a^3	> 26.5	n/a^3
96	> 26.5	n/a^3	> 26.5	n/a^3	> 26.5	n/a^3
	NOEC (ng/L)		NOEC (ng/L)		NOEC (ng/L)
72	26.5		26.5		26.5
96	26.5		26.5		26.5

¹ EC50, Ey50 and Er50 values and confidence limits, where possible, were calculated by non-linear regression, with replicate data (cell density, yield or growth rate) and day 0 measured test concentrations

² EyC50 values were only determined at 72 and 96 hours as per protocol

³ 95% confidence intervals could not be calculated with the data obtained

Conclusions:

The freshwater alga (Pseudokirchneriella subcapitata), was exposed to a geometric series of five concentrations of test article (AN-2) ranging from 1.50 to 26.5 ng/L (based on day 0 measured test concentration). There were no statistically significant treatment-related effects on cell density, yield and growth rate at concentrations equal or less than 26.5 ng/L. Consequently, the 72 and 96 hour NOECs for cell density, yield and growth rate were 26.5 ng/l (the highest concentration tested). EC50 values at 72 hours for the same effects were all greater than 26.5 ng/L.

Executive summary:

The freshwater alga(Pseudokirchneriella subcapitata), was exposed to a geometric series of five concentrations of test article (AN-2) ranging from 1.50 to 26.5  ng/L (based on day 0 measured test concentration). There were no statistically significant treatment-related effects on cell density, yield and growth rate at concentrations equal or less than 26.5 ng/L. Consequently, the 72 and 96 hour NOECs for cell density, yield and growth rate were 26.5 ng/l (the highest concentration tested). EC50 values at 72 hours for the same effects were all greater than 26.5 ng/L. 

Description of key information

Two standard algae study are available that confirm that the substance is not toxic to freshwater algae up to its solubility limit. Both the EC50 and the NOEC are above the solubility limit in the medium. 

Additional information

Two standard guideline algae studies are available. An older study was only conducted with oversaturated nominal concentrations of 1000 mg/l, while the recent study confirmed the test concentrations analytically and applied test concentrations at the solubility limit. No effects on algae growth were observed at the maximum achievable concentration and it can be concluded that the substance did not have acute or chronic effects in the test system up to its solubility limit.