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

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 19 to April 9, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Followed existing guideline and GLP requirements

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)

Qualifier:

according to guideline

Guideline:

other: ASTM Standard E 1193-97

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples were collected from each treatment group
and controls five days prior to the start of the test. Water samples were also
collected from alternating replicate test chambers in each group at the beginning of the test, at weekly intervals during the test and at the end of the test to measure test substance concentration. Samples of the stock solutions being delivered to the diluters were collected on Day -11 to confirm stock concentrations. Test solutions were also collected on Day 22 to confirm the concentration of the 1.6 ng/L treatment level when the analytical data on Day 21 for this group was suspect.
Radioactive stock solutions were transferred to scintillation vials and
analyzed by liquid scintillation counting (LSC), using a Packard Liquid
Scintillation Analyzer. Test waters were extracted twice with dichloromethane, evaporated to dryness, and reconstituted with acetonitrile. Extracts were then analyzed by LSC.

Vehicle:

yes

Details on test solutions:

Individual stock solutions for each of the six concentrations were prepared once during the study. Each stock solution was
prepared by mixing a calculated amount of radiolabelled test article into HPLC-grade dimethylformamide (DMF) at a nominal concentration of 0.064, 0.124, 0.252, 0.52, 1.0, and 2.0 ug/mL. Solutions were inverted to mix. Stock solutions up to 1.0 ug/mL were clear, and the 2.0 ug/mL solution had a slight purple tint. Stock solutions were stored refrigerated in amber bottles, and aliquots placed in the syringe pump every 4 to 7 days as needed during the study. The stock solutions were injected into the diluter mixing chambers at a rate of 2.5 uL/minute, where they were mixed with dilution water delivered at a rate of 100 mL/minute to achieve the desired test concentrations. Negative control was dilution water only. Solvent control was prepared by delivering HPLC-grade DMF to the appropriate mixing chamber. The concentration of DMF in the solvent control and all treatment groups was 0.025 mL/L.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Source: Wildlife International, Ltd.
- Age of parental stock : Culture stocked maintained continuously at laboratory. Neonates chosen for test were < 24 hours old
- Feeding during test: Yes
- Food type: During culture and testing, daphnids were fed a mixture of yeast, cereal grass media, and trout chow (YCT), as well as a suspension of the freshwater green algae (Pseudokirchneriella subcapitata).
- Amount: At each feeding, each test chamber was fed 0.75 mL of YCT and 1.5 mL of algae, amounting to about 0.7 mg C/daphnid/day. This exceeds the OECD guideline, but was fed in order to maintain sufficient feed in the flow-through system to support acceptable reproduction rates.
- Frequency: Daphnids were fed two or three times per day through Day 7 of test and then four times daily until the end of the test.

ACCLIMATION
- Acclimation period: Adults held at least 17 days prior to test, and had produced at least 1 brood
- Acclimation conditions (same as test or not): Yes
- Health during acclimation (any mortality observed): No observations of stress, mortality, or disease

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

Moderately hard.
Values over 4 weeks prior to test:
Mean hardness (mg/L as CaCO3) 141, range of 140-144. N = 4
Alkalinity (mg/L as CaCO3) 178, range 176-178

Test temperature:

Values during test conditions: manual. Temperature during the test were within the 20 degrees C+/- 1 degree set for the test
Day O values: range 19.7 to 19.9 degrees C
Day 21 values: range 20.1 to 20.4 degrees C

Continuous values from one control test chamber ranged from 19 to 20 degrees C, measured to nearest degree

pH:

Values over 4 weeks prior to test:
pH mean 8.1, range 8.0 - 8.1
pH mean over test: 8.0 to 8.2

Dissolved oxygen:

Values during test: Dissolved oxygen concentrations remained >/= 62% (5.6 mg/L)
Day O values: range 7.6 to 8.5 mg/L
Day 21 values: range 6.3 to 8.2 mg/L

Salinity:

Freshwater

Nominal and measured concentrations:

Nominal Mean Measured
Negative control < LOQ
Positive Control < LOQ
1.6 ng/l 1.4 ng/l
3.1 ng/l 2.4 ng/l
6.3 ng/l 5.5 ng/l
13 ng/l 12 ng/l
25 ng/l 21 ng/l
50 ng/l 43 ng/l

Details on test conditions:

TEST SYSTEM
- Test vessel: Test chambers: 9L glass aquaria. Test compartments 300 mL glass beakers
- Material, size, headspace, fill volume: Aquaria filled with 8L test water. Test chambers had two holes on opposite sides to allow test solution to flow in or out. depth of water in representative compartment was 8.7 cm, while depth of water in representative chamber was 18.5 cm.
- Type of flow-through : continuous diluter.
- Renewal rate of test solution (frequency/flow rate): 9 volume additions per day
- No. of organisms per vessel: 5 daphnids in each of two compartments per chamber. Two chambers per replicate. Total of 20 daphnids per condition.
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2


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. Specific conductance, hardness, alkalinity and pH of well water were monitored during the 4 week period prior to the study. Periodic analysis of the well water for selected organic and inorganic constituents was conducted.
- Total organic carbon: less than 1 mg/L at test intiation and termination in dilution water
- Particulate matter: filtered to less than 0.45 um
- Metals: below limits of detection when analyzed 12/28/2011
- Pesticides: below limits of detection when analyzed 12/28/2011
- Alkalinity: 176- 178 mg/L as CaCO3 in negative control during test; 178 to 180 in high dose group during test
- Ca/mg ratio: 34.9/13.2 mg/L in dilution water prior to test
- Conductivity: 354 uS/cm, range 335-367 prior to test; 361 to 375 in negative control during test; 365 to 381 in high dose group during test
- Salinity: freshwater
- Culture medium different from test medium: no
- Intervals of water quality measurement: TOC in dilution water at intiation and end of test. pH, hardness, alkalinity and specific conductance at beginning of test, weekly and at end. Dissolved oxygen at beginning, 3 times weekly and end of test.

OTHER TEST CONDITIONS
- Photoperiod: 16 hour light/8 dark
- Light intensity: 171 lux at water surface at test initionation

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Results used to determine the conditions for the definitive study: Previous acute toxicity studies, Measured water solubility of substance,

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 43 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: not calculated

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 43 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: not calculated

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 43 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: not calculated

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 43 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth

Remarks on result:

other: not measured

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 43 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: not calculated

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 43 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: not calculated

Details on results:

- Mortality of parent animals: Survival in the negative control and solvent control groups were 75 and 95% respectively. Control group data was pooled. Survival of the 1.4, 2.4, 5.5, 12, 21 and 43 ng/L groups were 90, 90, 100, 95, 90 and 90%
- No. of offspring produced per day per female: Adult daphnids in the negative and solvent control groups produced an average of 8.1 and 8.7 live young per reproductive day. Adults in the 1.4, 2.4, 5.5, 12, 21 and 43 ng/L groups prodeuced 8.9, 6.7, 8.1, 6.9, 7.7, and 8.3 offspring per reproductive day.
- Body length and weight of parent animals: Daphnids in the negative and solvent control groups averaged 4.6 and 4.5 mm in length and 0.63 and 0.69 mg in dry weight respectively. Daphnids in the 1.4, 2.4, 5.5, 12, 21 and 43 ng/L groups had mean lengths of 4.6, 4.3, 4.4, 4.4, 4.4, and 4.5 mmg respectively, and mean weights of 0.76, 0.60, 0.74, 0.76, 0.63, and 0.60 mg respectively. The values in the 2.4, 5.5, 12, and 21 ng/L gropus were statistically reduced, but, the reductions did not fall into a dose response pattern, and were not considered biologically significant. Mean length of daphnids historically of this age and at this laboratory ranged from 3.6 to 6.3 mm.
- Type and number of morphological abnormalities: Daphnids in treatment groups that survived generally appeared normal. During the test, pale, small, and/or lethargic daphnids were occasionally observed in the 1.4, 2.4, 12, 21, and 43 ng/l groups. The numbers of sublethal effects seen in the 1.4, 12, and 21 ng/L groups were infrequent. At test termination, about 33% of surviving first generation daphnids in the high dose group were pale compared to the control group. All surviving daphnids in the 2.4 and 5.5 ng/l gropus were normal in appearance at termination.

Sumary of Survival, Reproduction, and Growth of Daphnia magna exposed to Radiolabelled AN-2 for 21 days

Mean Measured Concentration (ng/L)	% Adult Survival^1,2	Mean Neonates per Reproductive Day +/- STD^1,3	Mean Length +/- STD (mm)	Mean Dry Weight +/- STD^1 (mg)
Negative Control	75	8.1 +/-0.53	4.6 +/- 0.06	0.63 +/- 0.11
Solvent Control	95	8.7 +/- 1.3	4.5 +/- 0.05	0.69 +/- 0.033
Pooled Control	85	8.4 +/- 1.0	4.5 +/- 0.05	0.66 +/- 0.074
1.4 ng/l	90	8.9 +/- 1.2	4.6 +/- 0.06	0.76 +/- 0.10
2.4 ng/l	90	6.7 +/- 1.1	4.3 +/- 0.10^*,4	0.60 +/- 0.013
5.5 ng/l	100	8.1 +/- 0.74	4.4 +/- 0.08^*,4	0.74 +/- 0.046
12 ng/l	95	6.9 +/- 1.1	4.4 +/- 0.00^*,4	0.76 +/- 0.095
21 ng/l	90	7.7 +/- 1.7	4.4 +/- 0.05^*,4	0.63 +/- 0.055
43 ng/l	90	8.3 +/- 1.6	4.5 +/- 0.13	0.60 +/- 0.039

* Statistically significant difference in mean total from pooled control (Dunnett’s one-tailed test, p</=0.05)

¹ No significant difference in survival (Fisher’s Exact Test, p > 0.05), reproduction or mean dry weight (Dunnett’s one-tailed test, p > 0.05)

² 21-day EC50 survival: > 43 ng/L

³ 21-day EC50 for reproduction: > 43 ng/L

⁴ Statistically significant differences in mean total length noted at 2.4, 5.5, 12 and 21 ng/L not considered biologically significant

Validity criteria fulfilled:

yes

Conclusions:

NOEC for test article in a 21 day Daphnia magna flow through test was 43 ng/L., the highest achievable concentration. 21 day EC 50 values were > 43 ng/L for reproduction and immobility.

Executive summary:

The cladoceran, Daphnia magna, was exposed to test article (AN-2) at mean measured concentrations of 1.4, 2.4, 5.5, 12, 21, and 43 ng/L under flow through conditions for 21 days. There were no statistically significant treatment-related effects on survival, reproduction or growth at concentrations equal or less than 43 g/L. In addition, the mean length of the daphnids in the 2.4, 5.5, 12, and 21 ng/L groups were within the ranges of the mean length of daphnids in the historical control data (3.6 to 6.3 nm). Consequently, the NOEC for growth (length and dry weight) and reproduction was 43 ng/L and the LOEC was greater than 43 ng/L, the highest achievable test concentration.

Description of key information

A 21-day daphnia reproduction study conducted according to recent test guidelines and under GLP did not reveal any adverse effect at the maximum achievable test concentration that was analytically verified.

Additional information

A 21-day daphnia study was conducted with the substance that due to its low water solubility did not reveal acute toxicity to aquatic organisms. The study did not reveal any adverse effects on daphnia growth or reproduction up to the highest achievable concentration, its solubility limit in the medium that was analytically verified in the test. The substance does not reveal chronic toxicity in the test system up to its solubility limit.