Dibenzylbenzene, ar-methyl derivative

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1995-05-15 to 1995-05-19

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: WAF
- Sampling method: directly taken from the test solution, for stability control the sample was maintained for 24 hours
- Sample storage conditions before analysis: the stability control sample was maintained under test conditions without fish, no furhter details mentioned

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of stock solution: 1 g test substance/l tap water, stirring for approx. 18 h, thereafter filtration. The test solutions were freshly prepared every day.
- Controls: yes, tap water
- Evidence of undissolved material (e.g. precipitate, surface film, etc): not applicable

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebrafish
- Strain: Brachydanio rerio
- Source: West Aquarium, Bad Lauterberg, Germany
- Age at study initiation (mean and range, SD): not mention
- Length at study initiation (length definition, mean, range and SD): 3 ± 0.5 cm, no further details mentioned
- Weight at study initiation (mean and range, SD): approx. 0.4 g, no further details mentioned
- Method of breeding: not mentioned
- Feeding during test: none


ACCLIMATION
- Acclimation period: 14 days
- Acclimation conditions (same as test or not): The fish were kept in 200 l earthenware basins, or in 300 l glass aquariums in flow rate.
- Type and amount of food: TetraMin, administration in an amount of approx. 1 % of body weight
- Feeding frequency: daily
- Health during acclimation (any mortality observed): The animals showed a normal behaviour pattern at the beginning of the test and were free from visible diseases. The mortality rate during the holding phase was <5% seven days before the beginning of the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

none

Hardness:

approx. 12 °dH

Test temperature:

19 - 20 °C

pH:

7.8 - 8.4

Dissolved oxygen:

84 - 109 % O2 saturation

Salinity:

not mentioned

Nominal and measured concentrations:

Nominal: WAF
Measured: 0.32 mg/L (arithmetic mean of freshly prepared test solutions)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): not mentioned
- Material, size, headspace, fill volume: 20 l, 10 l fill volume, no further details mentioned
- Aeration: continously, no further details mentioned
- Renewal rate of test solution (frequency/flow rate): daily renewal
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.4 g fish/L


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: drinking water, Gelsenwasser AG, Germany
no further details mentioned
- Intervals of water quality measurement: not mentioned

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light, 8 hours darkness
- Light intensity: not mentioned


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality after 24, 48, 72 and 96 hours; pH, temperature, O2 saturation at the end of the test


TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable (limit test)
- Range finding study: not performed

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.32 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

0.32 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: nothing mentioned
- Observations on body length and weight: not performed
- Other biological observations: none
- Mortality of control: 0 % after 96 h
- Other adverse effects control: none
- Abnormal responses: nothing mentioned
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: nothing mentioned
- Effect concentrations exceeding solubility of substance in test medium: not applicable

Results with reference substance (positive control):

no reference substance tested

Reported statistics and error estimates:

no statisitcs performed

Sublethal observations / clinical signs:

concentration [mg/L]	No. of fish at start of study	observation period
		24 h		48 h		72 h		96 h
		No. dead	mortality [%]	No. dead	mortality[%]	No. dead	mortality[%]	No. dead	mortality[%]
control	10	0	0	0	0	0	0	0	0
0.32	10	0	0	0	0	0	0	0	0

Validity criteria fulfilled:

yes

Conclusions:

No toxicity was observed at the tested concentrations

Executive summary:

An acute toxicity study with Dibenzyltoluene was performed using WAF conditions. Under these test conditions, no adverse effect was observed after 96h of exposure.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 1990-02-06 to 1990-02-20

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: A surfactant was used in the study to disperse the substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 204 (Fish, Prolonged Toxicity Test: 14-day Study)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.10; 0.32; 0.60 and 1.0 mg/L
- Sampling method: layer chromatography
- Sample storage conditions before analysis: not mentioned

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 1 g test substance + 1 g vehicle/L, no further details mentioned
- Controls: yes, blank and vehicle control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Castor oil, ethoxylated (40 EO)
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): stock solution: 1 g/L; final test solutions: same as test material, vehicle control: 0.10; 0.32; 0.60 and 1 mg/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc): nothing mentioned

Test organisms (species):

Leuciscus idus melanotus

Details on test organisms:

TEST ORGANISM
- Common name: Golden orfe
- Strain:Leuciscus idus melanotus
- Source: Fischzucht Eggers, Hohenwestedt, Germany
- Age at study initiation (mean and range, SD): not mentioned
- Length at study initiation (length definition, mean, range and SD): 6.3 to 7.5 cm, no further details mentioned
- Weight at study initiation (mean and range, SD): not mentioned
- Method of breeding: not mentioned
- Feeding during test: two times per day (1 % TetraMin per average weight)

ACCLIMATION
- Acclimation period: 14 days
- Acclimation conditions (same as test or not): The fish were kept in dechlorinated tap water in 36 L glass aquariums in flow rate. After supply the fish were once treated for 1 hour with ZEPHiROL (20 mg/mL), temperature 22 +/- 2 °C
- Type and amount of food: TetraMin, administration in an amount of approx. 1 % of body weight
- Feeding frequency: daily
- Health during acclimation (any mortality observed): only healthy animals used for the test

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

14 d

Post exposure observation period:

no

Hardness:

not mentioned

Test temperature:

21.6-22.2 °C

pH:

7.9-8.2

Dissolved oxygen:

7.6-8.0 mg/mL

Salinity:

230 mg CaCO3 = 12.8 °dH

Nominal and measured concentrations:

nominal/measured [mg/mL]
0.10/0.08 +/- 0.02
0.32/0.24 +/- 0.12
0.60/0.39 +/- 0.12
1.00/0.46 +/- 0.14

Details on test conditions:

TEST SYSTEM
- Test vessel: glass aquarium
- Type (delete if not applicable): open
- Material, size: glass
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): not mentioned
- Renewal rate of test solution (frequency/flow rate): 20 L/h
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 1
- Biomass loading rate: not mentioned

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: not mentioned
- Ca/mg ratio: 230 mg CaCO3
- Culture medium different from test medium: no
- Intervals of water quality measurement: not mentioned

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 14/10 h
- Light intensity: not mentioned

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not mentioned
- Justification for using less concentrations than requested by guideline: not mentioned
- Range finding study: not performed
- Test concentrations: 0.10; 0.32; 0.60 and 1.0 mg/L

Reference substance (positive control):

no

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

0.46 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Validity criteria fulfilled:

not specified

Conclusions:

No effect was observed until the highest tested concentration of 0.46 mg/L measured (1.0 mg/L nominal). The mortalities in some treatment groups are not substance specific. No signs of toxicity or abnormal behaviour observed.

Executive summary:

No effect was observed until the highest tested concentration of 0.46 mg/L measured (1.0 mg/L nominal). The mortalities in some treatment groups are not substance specific. No signs of toxicity or abnormal behaviour observed.

Reference 3

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

1990-08-21 until 1990-08-15

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: GLP guideline study but variable concentrations of surfactant vehicle. Concentrations tested well above solubility

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

GLP compliance:

yes

Specific details on test material used for the study:

The purity of the material used for the test was lower than the purity of the actual material. It contained ca. 26% of isomers of dimethyl tribenzenes. However, based on comparable basic structure of the impurities and the assumption that any toxic effects would increase with higher molecular weight and lipophilicity, we regard that the studies conducted with the lower purity material being "worst case" and can be used for the evaluation of the actual product.

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control; vehicle control; 0.25; 0.35; 0.5; 0.7; 1.0 mg/L (after 0 h and 96 h post exposure) and spiked water samples of 0.3; 1 mg/L Marlotherm S
- Sampling method: not mentioned
- Sample storage conditions before analysis: not mentioned

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Procedure of stock solution preparation: The test substance was added dropwise to the vehicle. While stirring the dissolved test material, reconstituted water was added.
- Controls: yes, reconstituted water and vehicle control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Ricinoleicacid ethoxylate
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)):
Stock solution: 50 mL/L
Final test solutions: 0.0115 - 0.0248 mL/L
Vehicle control: 0.0245 m//L
- Evidence of undissolved material (e.g. precipitate, surface film, etc): nothing mentioned

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Strain: Salmo gairdneri
- Source: Forellenhof Fredelsloh, Mohringen, Germany
- Age at study initiation (mean and range, SD): not mentioned
- Length at study initiation (sample consisting of 10 fish): 3.7 ± 0.283 cm/fish
- Weight at study initiation (sample consisting of 10 fish): 0.464 ± 0.054 g/fish
- Method of breeding: not mentioned
- Feeding during test: none


ACCLIMATION
- Acclimation period: > 30 d
- Acclimation conditions (same as test or not): not the same
- Type and amount of food: Rheinkrone, daily approx. 2% of mean fish weight
- Feeding frequency: 5 times a week except the last day before starting the test
- Health during acclimation (any mortality observed): 0% mortality during the period of 7 d before start of the test

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

none

Hardness:

178 / 213 mg/L CaCO3 min/max

Test temperature:

14.6 - 15.5 °C (mean ± SD: 15.01 ± 0.20 °C)

pH:

7.5 - 7.9 (mean ± SD: 7.82 ± 0.09)

Dissolved oxygen:

8.9 - 10.7 mg/L (mean ± SD: 9.59 ± 0.56 mg/L)

Salinity:

294 mg/L CaCl2 x 2 H2O
123.25 mg/L MgSO4 x 7 H2O
64.75 mg/L NaHCO3
5.75 mg/L KCl

Nominal and measured concentrations:

Nominal: control / vehicle control / 0.25 / 0.35 / 0.48 / 0.70 / 1.0 mg/L
Measured: The analytically determined actual concentrations of the test material at the beginning of the study were found to be within the range of 72% and 99% with an average of 85.4%. The analytically determined stability of the test material after 96 hours of exposure were found to be within the range of 101% and 137% of the actual values with an average of 117.8%.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, fill volume: glass, 22 L (height: 26 cm, length: 34.5±0.5 cm, width: 21.5±0.5 cm), 10 L
- Aeration: the reconstituted water was aerated for several hours, no further details mentioned
- Type of flow-through: A membrane pump was connected to the tanks(with stock solution of the test material, the solution for the vehicle control, the reconstituted water) and delivered the solution against a pressure keeping valve, across a distributor towards a magnetic valve. From there the stock solution and reconstituted water were added simultaneously to mixing bottles (Woulff Bottles). The solutions were stirred and the test solutions transferred into the test vessels, where an overflow kept the test solutions at constant levels.
- Renewal rate of test solution (frequency/flow rate): 99.71 ± 2.84 L/d
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 1
- Biomass loading rate: 0.464 g/L


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Four solutions (A, B, C, D) were prepared in deionized water with a conductivity of < 1 µS/cm
A) 11.76 g/L CaCl2 x 2 H2O
B) 4.93 g/L MgSO4 x 7 H2O
C) 2.59 g/L NaHCO3
D) 0.23 g/L KCl
25 mL of each of the four solutions were mixed and made up to 1 L with deionized water and aerated for several hours (= reconstitued water).
- Culture medium different from test medium: no
- Intervals of water quality measurement: not mentioned


OTHER TEST CONDITIONS
- Adjustment of pH: not adjusted
- Photoperiod: 8/16 h dark/light
- Light intensity: 100 Lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Mortality of fish after 0, 3, 6, 24, 48, 72 and 96 h; pH, O2 saturation, temperature (35 measurements)


TEST CONCENTRATIONS
- Spacing factor for test concentrations: not mentioned
- Range finding study: not performed, toxicity data provided by the sponsor were used to set the concentration levels for the definitive test

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

0.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: not observed
- Observations on body length and weight: not determined
- Other biological observations: none
- Mortality of control: 0%
- Other adverse effects control: none
- Abnormal responses: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: no

The lowest concentration causing 100% mortality within the period of the test could not be determined.

Sublethal observations / clinical signs:

concentration [mg/L]	No. of fish at start of study	observation period
		3 h		6 h		24h		48 h		72 h		96 h
		No. dead	mortality[%]	No. dead	mortality[%]	No. dead	mortality[%]	No. dead	mortality[%]	No. dead	mortality[%]	No. dead	mortality[%]
control	10	0/10	0	0/10	0	0/10	0	0/10	0	0/10	0	0/10	0
vehicle control	10	0/10	0	0/10	0	0/10	0	0/10	0	0/10	0	0/10	0
0.25	10	0/10	0	0/10	0	0/10	0	0/10	0	0/10	0	0/10	0
0.35	10	0/10	0	0/10	0	0/10	0	0/10	0	0/10	0	0/10	0
0.5	10	0/10	0	0/10	0	0/10	0	0/10	0	0/10	0	0/10	0
0.7	10	0/10	0	0/10	0	0/10	0	0/10	0	0/10	0	0/10	0
1.0	10	0/10	0	0/10	0	0/10	0	2/10	20	2/10	20	2/10	20

Validity criteria fulfilled:

yes

Conclusions:

In the highest test material concentration tested the mortality was 20% at the end of the test period. No statistical analysis were performed.

Executive summary:

An acute toxicity study was performed with dibenzyltoluene substance under flow-through conditions and shows 20% of mortality at the end of the exposure period (96h) only at the highest tested concentration.

Reference 4

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

1990-08-13 to 1990-08-17

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Concentrations well above water solubility limit, no information on vehicle used.

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

GLP compliance:

yes

Specific details on test material used for the study:

The purity of the material used for the test was lower than the purity of the actual material. It contained ca. 26% of isomers of dimethyl tribenzenes. However, based on comparable basic structure of the impurities and the assumption that any toxic effects would increase with higher molecular weight and lipophilicity, we regard that the studies conducted with the lower purity material being "worst case" and can be used for the evaluation of the actual product.

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.52, 0.95, 1.7, 3.2 mg/L
- Sampling method: not mentioned
- Sample storage conditions before analysis: not mentioned

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: not mentioned
- Controls: yes, dilution water and vehicle control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not mentioned
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): highest concentration in test solution: 5 mg/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc): nothing mentioned

Test organisms (species):

Leuciscus idus melanotus

Details on test organisms:

TEST ORGANISM
- Common name: Golden orfe
- Strain: Leuciscus idus melanotus HECKEL
- Source: Fischzucht Eggers, Hohenwestedt, Germany
- Age at study initiation (mean and range, SD): not mentioned
- Length at study initiation (length definition, mean, range and SD): 6 ± 2 cm, no further details mentioned
- Weight at study initiation (mean and range, SD): not mentioned
- Method of breeding: not mentioned
- Feeding during test: none


ACCLIMATION
- Acclimation period: 14 days
- Acclimation conditions (same as test or not): The fish were kept in dechlorinated tap water in 200 l earthenware basins, or in 300 l glass aquariums in flow rate. After supply the fish were once treated for 1 hour with ZEPHIROL (1:50000).
- Type and amount of food: TetraMin, administration in an amount of approx. 3 % of body weight
- Feeding frequency: not mentioned
- Health during acclimation (any mortality observed): only healthy animals were used for the test, no further details mentioned

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

none

Hardness:

15 °dH

Test temperature:

21 +/- 1 °C

pH:

7.7 - 8.1

Dissolved oxygen:

7.9 - 9.2 mg/L

Salinity:

not mentioned

Nominal and measured concentrations:

Nominal: 0.52, 0.95, 1.7, 3.2 mg/L
Measured: 0.6, 0.9, 1.9, 3.7 mg/L
The nominal concentrations were used for evaluation

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): not mentioned
- Material, size, headspace, fill volume: 35 L volume, no further details mentioned
- Type of flow-through (e.g. peristaltic or proportional diluter): not mentioned
- Renewal rate of test solution (flow rate): 10 L/h
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: not mentioned


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated drinking water
no further details mentioned
- Intervals of water quality measurement: not mentioned

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light, 8 hours darkness
- Light intensity: not mentioned


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality after 24, 48, 72 and 96 hours; pH, temperature, O2 saturation at the end of the test


TEST CONCENTRATIONS
- Spacing factor for test concentrations: not mentioned
- Range finding study: not mentioned

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 3.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: nothing mentioned
- Observations on body length and weight: not determined
- Other biological observations: Up to a concentration of 3.2 mg/L no acute toxicity was observed
- Mortality of control: 0%
- Other adverse effects control: none
- Abnormal responses: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: not applicable

Results with reference substance (positive control):

no reference substance tested

Reported statistics and error estimates:

no statisitcs performed

Sublethal observations / clinical signs:

Table: Mortality data

concentration[mg/L]	No. of fishat start of study	observation period
		24 h		48 h		72 h		96 h
		No. dead	mortality [%]	No. dead	mortality[%]	No. dead	mortality[%]	No. dead	mortality[%]
control	10	0	0	0	0	0	0	0	0
vehicle control	10	0	0	0	0	0	0	0	0
0.52	10	0	0	0	0	0	0	0	0
0.95	10	0	0	0	0	0	0	0	0
1.7	10	0	0	0	0	0	0	0	0
3.2	10	0	0	0	0	0	0	0	0

Validity criteria fulfilled:

yes

Conclusions:

No acute toxicity was observed

Executive summary:

An acute toxicity study was performed with dibenzyltoluene substance under flow-through conditions. No toxicity was observed until the end of the exposure period (96h).

Reference 5

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 11 March 2010 to 10 March 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

For the purpose of chemical analysis, duplicate aliquots (ca 20 mL) were removed from test and control WAF at the commencement of the test (0 h) and at 72 h as `fresh¿ solution and at 24 and 96 h from test and control tanks as `expired¿ solution.

Vehicle:

no

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Zebra fish (Brachydanio rerio) in the size range 1-3 cm were used for this test and were received from Water Wonders, Edinburgh on 17 July 2009. All fish were in good health and free from any apparent malformation.

All fish were acclimatised to laboratory conditions for a minimum of 14 days prior to commencement of the test. During holding, the fish were fed daily on a suitable standard fish diet. The fish were not fed for a period of 24 h prior to test commencement or throughout the duration of the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

no

Hardness:

3.6 mg CaCO3/L

Test temperature:

22.1 - 23.0 °C

pH:

Fresh medium: 6.6
Prior to medium renewal: 6.3

Dissolved oxygen:

Fresh medium: 83 - 92 % air saturation value
Prior to medium renewal: 60 - 63 % asv

Nominal and measured concentrations:

WAF were prepared with nominal concentrations of 1 mg/L. Measured concentrations were either below the lowest point on the curve or are equivalent to the background concentration (45 - 69 µg/L)

Details on test conditions:

TEST SYSTEM
Tanks of ca 20 L capacity of moulded glass construction and sealed with glass lids to prevent a head-space of air in the tank
One tank was prepared at the initial loading rate of 1 mg dibenzyltoluene/L and one control, containing control WAF.
Seven zebra fish were added to each tank within 30 min of preparation.
Fish were transferred to freshly prepared test solutions at 24 h intervals.

TEST MEDIUM / WATER PARAMETERS
Reconstituted freshwater (RFW) prepared at Charles River, with high grade salts and reverse osmosis grade water was used during the holding and testing phases of the study. The salts were dissolved in a volume of deionised water (500 L) and this solution was metered into a flow of deionised water, to produce RFW according to the formula recommended by OECD Guideline 203.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 50 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: no effect at solubility limit, lower than analytical LOQ ca. 50 µg/L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

50 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: no effect at solubility limit, lower than analytical LOQ ca. 50 µg/L

Details on results:

The fish in the limit test were observed after 1, 4, 24, 48, 72 and 96 h.
At the termination of the test, the length (from the tip of the snout to the caudal peduncle) and weight of each surviving fish was recorded.
Analytical samples taken at 0 and 24 h and at 72 and 96 h indicated that concentrations of dibenzyltoluene were below the limit of detection. Measured concentrations were, in all cases, equivalent to background concentration and equal to the values obtained for the control sample analysis.

All test solutions appeared clear and colourless throughout the test period. No precipitation of dibenzyltoluene was observed.

There was no mortality recorded during the test). All fish appeared active and healthy throughout.

The length and weight of fish after 96 h exposure were measured. Fish were within the range 26-30 mm in length and 0.258-0.417 g in weight.

Reported statistics and error estimates:

Statistical analysis was not conducted on the data as there was no mortality.

Conclusions:

The 96 h LC50 of dibenzyltoluene to zebra fish is concluded to be greater than the maximum solubility of dibenzyltoluene in water, under the conditions of the test. The NOEC is concluded to be the maximum solubility of dibenzyltoluene in test water.

Executive summary:

The acute toxicity of dibenzyltoluene to zebra fish (Brachydanio rerio)was assessed by conducting a limit test at the maximum solubility of dibenzyltoluene under test conditions. The test was conducted over a 96 h period under semi-static conditions, in accordance with OECD (1992) Guideline 203, following procedures in OECD (2000) Guidance Document No. 23 on Testing Difficult Substances.

Test solutions were prepared as Water Accommodated Fractions (WAF¿s) from an initial loading rate of 1 mg dibenzyltoluene/L. The method of test solution preparation was selected to maximise the solubility of dibenzyltoluene under test exposure conditions, while minimising exposure to insoluble fractions. A control of untreated test water was also included.

One test tank (containing dibenzyltoluene WAF) and one control tank (containing untreated control WAF) were prepared and 7 zebra fish were added to each. Fish were transferred to freshly prepared solutions at 24 h intervals.

No mortality or other adverse effects were noted throughout the test period.

Analytical samples taken at 0 and 24 h and at 72 and 96 h indicated that concentrations of dibenzyltoluene were below the limit of detection. Results throughout this report are therefore based on initial loading rate.

The 96 h LC₅₀of dibenzyltoluene to zebra fish is concluded to be greater than the maximum solubility of dibenzyltoluene in water, under the conditions of the test. The no observed effect concentration (NOEC) is concluded to be the maximum solubility of dibenzyltoluene in test water.

Reference 6

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: non standard, range-finding test

Qualifier:

according to guideline

Guideline:

other: AFNOR T90-303

Deviations:

yes

GLP compliance:

no

Analytical monitoring:

no

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

no data

Test type:

static

Water media type:

not specified

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

no

Hardness:

no data

Test temperature:

no data

pH:

no data

Dissolved oxygen:

no data

Salinity:

no data

Nominal and measured concentrations:

nominal

Reference substance (positive control):

not specified

Duration:

72 h

Dose descriptor:

LC0

Effect conc.:

> 400 mg/L

Details on results:

No mortality was observed at 24, 48 and 72 hours in any of the experiments up to 400 mg/l nominal concentration of UGILEC 102.
With UGILEC 101 mortality was observed: CL(I)50-24h = 3.8 mg/L and CL(I)50-48h = 3.5 mg/L

Validity criteria fulfilled:

not specified

Conclusions:

Very few information about test conditions, no mortality of Danio rerio was observed in 72 h with dibenzyltoluene up to very high nominal concentration 400 mg/L (from stock solutions in acetone).

Executive summary:

Very few information about test conditions, no mortality of Danio rerio was observed in 72 h with dibenzyltoluene up to very high nominal concentration 400 mg/L (from stock solutions in acetone). Under similar conditions benzyltoluene showed acute toxicity CL(I)50 -48h = 3.5 mg/L.

Reference 7

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: use of solvent for increasing bioavailability well above realistic concentrations (water solubility) ; test substance is a mixture of benzyltoluene and dibenzyltoluene

Qualifier:

according to guideline

Guideline:

OECD Guideline 204 (Fish, Prolonged Toxicity Test: 14-day Study)

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: stock solution of substance in acetone and dispersion in the medium (flow -through 1 renewal every 2 hours)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 1 ml/l
- Evidence of undissolved material (e.g. precipitate, surface film, etc): surface film at high concentrations

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

no data

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

14 d

Hardness:

no data

Test temperature:

23°C

pH:

6.9 - 7.6 daily measurements

Dissolved oxygen:

> 60% saturation
fresh solutions 7.5 - 7.6 mgO2/L, in test vessels : 5.2 - 6.6 mgO2/L, daily measurements

Salinity:

no data

Nominal and measured concentrations:

nominal concentrations: 1 - 2.5 - 3.5 - 5 - 10 - 15 - 20 - 30 -40 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): no data
- fill volume: 2 litres
- Aeration: no data
- Type of flow-through (e.g. peristaltic or proportional diluter): test substance in acetone is dispersed in test medium, which is pumped into test vessels
- Renewal rate of test solution (frequency/flow rate): 1 L/h for 2 L test vessel capacity
- No. of organisms per vessel: no data
- No. of vessels per concentration (replicates): no data
- No. of vessels per control (replicates): no data
- No. of vessels per vehicle control (replicates): no data
- Biomass loading rate: no data


TEST MEDIUM / WATER PARAMETERS
no data


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: no data

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
mortality, every working day

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 1 - 2.5 - 5 - 25 - 50 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

14 d

Dose descriptor:

LC100

Effect conc.:

10 mg/L

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

2.53 mg/L

Details on results:

- Mortality of control: no mortality in 14 days, no mortality in acetone control 1ml/l in 14 days
- Other adverse effects control: observation didn't report other clinical or behaviour sign of toxicity
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: test substance floating at high concentrations
- Effect concentrations exceeding solubility of substance in test medium: yes

Results with reference substance (positive control):

CL(I)50-24h = 269.9 mg/l in the validity range of French fish acute toxicity test AFNOR NF T 90-303 : 200 - 400 mg/l

Conclusions:

Although this study has been carried out under conditions that are not representative of reality, it can be inferred that no acute toxicity to Danio rerio is observed up to solubility limit.

Executive summary:

This fish prolonged acute toxicity test has been conducted with a mixture of benzyl- and dibenzyltoluene at concentrations well above solubility limit with the help of acetone as carrier. Concentration of acetone (1 ml/l) exceeded also admitted concentrations (0.1 ml/l).

In a preliminary test with a duration of 96h, it has been observed no mortality up to 2.5 mg/l. The same figure is obtained in the definitive test.

Description of key information

No acute toxicity (mortality or sublethal effects) is observed at solubility limit

Additional information

In a semi-static study, Scholz, 1995, didn't observed mortality of Danio rerio at a reported concentration of 0.32 mg/L. Exposure concentration was estimated by TLC, solutions were WAF. Few experimental details, as well as an exposure well above water solubility leads to conclude that this study is probably unreliable, but nevertheless that Dibenzylbenzene, ar-methyl derivative is probably not acutely toxic to fish.

Another study, on rainbow trout (Knacker et al, 1990), although being a GLP one, must be disregarded as it is made use of a surfactant, in varying concentrations, that is not recommended by OECD. Concentrations as high as 1 mg/L were tested (50 times higher than water solubility limit). 20% mortality was observed in 96 hours at that concentration.

A third study (Scholz, 1990c) on Golden Orfe has been carried out under flow-through conditions with the help of a vehicle whose identity is not available. Concentrations as high as 3.2 mg/L were tested; no mortality was observed.

Two other studies were reported (Lepailleur, 1981, 1983) regarding the prolonged acute toxicity (14 days) of Dibenzylbenzene, ar-methyl derivative to Danio rerio. The studies used high concentrations of acetone and the 14 day EC50 of 2.3 mg/L is not considered reliable for the same reasons as mentioned above.

An additional prolonged acute toxicity study was conducted under flow-through conditions with Golden Orfe (Scholz, 1990b). At none of the concentrations were there signs of toxicity or abnormal behavior that were attributable to dibenzyltoluene. The 14 day LC50 was >0.46 mg/L (measured concentration). As a surfactant was used to solubilize the Dibenzylbenzene, ar-methyl derivative, the resulting value is well above the level of water solubility.

Finally, the Companies in the consortium decided to commission a new study: using the OECD 203 protocol under semi-static conditions and under GLP conditions, it has been confirmed (Knight, 2010) that, at the highest exposure achievable with a Water Accommodated Fraction (yet lower than the analytical limit of quantification, 50 µg/L), there is no acute toxicity towards Danio rerio. The 96 h LC50 of dibenzyltoluene to zebra fish is concluded to be greater than the maximum solubility of Dibenzylbenzene, ar-methyl derivative in water, under the conditions of the test. The no observed effect concentration (NOEC) is concluded to be the maximum solubility of Dibenzylbenzene, ar-methyl derivative in test water.