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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1988-10-03 to 1988-10-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Guideline study (GLP Compliant)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 204 (Fish, Prolonged Toxicity Test: 14-day Study)
Deviations:
yes
Remarks:
See method description
Qualifier:
equivalent or similar to
Guideline:
other: ASTM E 729-80
Deviations:
yes
Remarks:
See method description
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Sampling method: 10mL were volumetrically transferred from the testing vessels using a pipet. All replicate treatment levels and the control solution were sampled and analyzed for radiolabelled test article prior to start of definitive exposure (test day 0) and on days 4, 8, 12, and 14. Samples were centrifuged within 30 minutes of sampling to remove suspended particles and adsorbed test article. Blind quality assurance samples were also prepared at each sampling period and remained with the set of exposure solution samples throughout the analysis.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution containing labeled and unlabeled 2,6-DTBP in acetone (4.18mg/ml) was delivered to a mixing chamber containing 2.510L of water. This mixing chamber was positioned over a magnetic stirrer with an ultrasonic water bath. The continuous stirring and sonication aided in the solublization of the test material.
- Controls: A vehicle control group at highest acetone concentration.
- Chemical name of vehicle (organic solvent): acetone
- Concentration of vehicle in test medium: 485uL/L in highest concentration (based upon discussions with US EPA)
- Evidence of undissolved material: None was observed (high dose required high level of acetone to maintain test article in solution)
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: Fathead Minnows
- Source: Springborn Life Sciences, Inc. (SLS lot #88A73)

- Feeding during test
- Food type: commercial pelleted food
- Amount: maintenance diet
- Frequency: test fish were not fed for the initial 4 days of the exposure. After day 4 they were fed every other day.

ACCLIMATION
- Acclimation period: 14 days
- Acclimation conditions: same as test
- Type and amount of food: commercial pelleted food
- Feeding frequency: ad libitum during acclimation
- Health during acclimation (any mortality observed): no mortalities were observed during two days prior to test initiation
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
14 d
Post exposure observation period:
Not applicable
Hardness:
26-32 mg/ml CaCO3
Test temperature:
21-22 degrees C
pH:
7.1-7.5
Dissolved oxygen:
7.3-10.4 mg/L
Salinity:
Specific conductance: 100-120 µm hos/cm
Nominal and measured concentrations:
Nominal concentrations:
-2.0, 1.3, 0.84, 0.55, 0.36 mg/L

Measure concentrations (mean, SD):
- 1.4 (0.099), 0.85 (0.061), 0.60 (0.064), 0.38 (0.028), 0.30 (0.047) mg/L

Nominal Concentration (mg/L): Measured Concentration (mg/L) (Day, replicate A or B)
2.0: 1.4(0,A), 1.5(0,B), 1.3(4,A), 1.3(4,B), 1.4(8,A), 1.5(8,B), 1.3(12,A), 1.4(12,B), 1.5(14,A), 1.5(14,B)

1.3: 0.77(0,A), 0.89(0,B), 0.75(4,A), 0.76(4,B), 0.88(8,A), 0.89(8,B), 0.86(12,A), 0.89(12,B), 0.89(14,A), 0.90(14,B)

0.84: 0.57(0,A), 0.63(0,B), 0.48(4,A), 0.55(4,B), 0.63(8,A), 0.61(8,B), 0.59(12,A), 0.60(12,B), 0.69(14,A), 0.69(14,B)

0.55: 0.39(0,A), 0.40(0,B), 0.32(4,A), 0.34(4,B), 0.38(8,A), 0.40(8,B), 0.40(12,A), 0.40(12,B), 0.40(14,A), 0.41(14,B)

0.36: 0.27(0,A), 0.27(0,B), 0.36(4,A), 0.41(4,B), 0.28(8,A), 0.28(8,B), 0.27(12,A), 0.27(12,B), 0.28(14,A), 0.31(14,B)

Solvent Control: <0.066(0,A, B), <0.066(4,A,B), <0.066(8,A, B) <0.065(12,A, B), <0.064(14,A), < 0.065(14,B)
Control: <0.066(0,A, B), <0.066(4,A,B), <0.066(8,A,B), <0.392(12,A)*, <0.065(12,B), <0.064(14,A,B)

*Measured concentration for this replicate indicates the presence of 2,6-DTBP in the control solutions. However, it is believed that the sample was contaminated during the analytical process.
Details on test conditions:
TEST SYSTEM
- Test vessel: Aquaria set in temperature control water baths
- Material, size, headspace, fill volume: Glass, 39x20x25cm, constant volume of 15L of water was maintained
- Aeration: Test solutions were not aerated
- Type of flow-through: Intermittent flow/proportional diluter. A dliuter constructed to deliver 500mL of solution per cycle
- Renewal rate of test solution (frequency/flow rate): 6.1 volume replacements per aquarium every 24 hours
- No. of organisms per vessel: 10
- 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
- Preparation of dilution water: The dilution water was obtained from the same source as the water which flowed into the fish holding tank.
-Total organic content: 4.3 ppm (historical)
- Pesticides: None
- Alkalinity: 22-24mg/L
- Ca/Mg ratio: 26-31 mg/L
- Conductivity: 100-120 umhos/cm
- Intervals of water quality measurement: every 24 hours


OTHER TEST CONDITIONS
- Photoperiod: 16 hours light/8 hours darkness
- Light intensity: 30-70 footcandles


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
% Mortality, toxic effects (loss of equilibrium, lethargy, and etc.) were observed and noted in the results of the study.


TEST CONCENTRATIONS
- Range finding study
- Test concentrations: Nominal concentrations of 4.0, 2.6, 1.7, 1.1, and 0.71mg/L 2,6-DTBP with end concentration of vehicle (acetone) of 82uL/L. Test concentrations as a mean of measured concentrations on days 0, 4, 8, 12, and 14 of the range finding study are 1.0, 0.66, 0.32, 0.24, and 0.20ug/L.
- Results used to determine the conditions for the definitive study: The above concentrations yielded only 5% mortality at the highest concentration. To achieve results needed to estimate the LD50 higher concentrations were needed. Preliminary trials performed demonstrated that at acetone concentrations of 500uL/L , higher levels of 2,6-DTBP can be solubilized and consistently maintained in exposure solutions. Based on discussion with the US EPA (who required the test) it was decided to exceed the recommended solvent limitations of 100uL/L and conduct test using a maximum vehicle concentration of 485uL/L. The nominal concentrations of 2,6-DTBP for the definitive test were set at 2.0, 1.3, 0.84, 0.55, and 0.36mg/L.

Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.4 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
dissolved
Basis for effect:
mortality
Duration:
7 d
Dose descriptor:
LC50
Effect conc.:
1.1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
dissolved
Basis for effect:
mortality
Remarks on result:
other: 95% CL (0.98-1.2)
Duration:
14 d
Dose descriptor:
LC50
Effect conc.:
1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
dissolved
Basis for effect:
mortality
Remarks on result:
other: 95% CL (0.85-1.4)
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
0.3 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
dissolved
Basis for effect:
behaviour
Details on results:
- Incidents in the course of the test which might have influenced the results: Deviations were not considered to affect the results of the study
1. During the study exposure solution samples were centrifuged for 15 minutes instead of being passed through a 0.45 micron filter prior to the analysis in order to remove debris. Centrifugation was sufficient to remove particles (0.45 microns) and other sorbed substances. Preliminary investigation established that filtering the exposure solutions removed soluble test material.
2. The concentration of solvent (vehicle, acetone) in the highest treatment level was 485 ul/L. This exceeded the 100 ul/L limit in the protocol. After consultation with US EPA (who required the study), it was decided to allow the exceedance in order to allow preparation and maintenance of consistent exposure concentrations to the test substance.
3. Daily sampling of exposure solution temperatures showed a range of 21 to 22 degrees C. The protocol required a range of 22 +/- 1 degree C. One replicate vessel was monitored continuously and showed a range of 21 to 24 degrees C.
4. The study protocol required measurement of TOC (total organic carbon) of the dilution water at study initiation, and that the value should not exceed 2 mg/L Analytical malfunction prevented this measurement. Historical values for TOC in dilution water averaged 4.3 ppm.


Summary of the LC50 values (95% confidence intervals) for each 24-hour interval during the 14-day exposure of fathead minnows (Pimephales promelas) to 2,6-DTBP.

Day 1-3 >1.4a
Day 4 1.4b
Day 5 1.4b
Day 6 1.3c(0.85-)
Day 7 1.1d(0.98-1.2)
Day 8 1.1d(0.98-1.2)
Day 9 1.1e(0.85 -1.4)
Day 10 1.0e(0.85-1.4)
Day 11 1.0e(0.85-1.4)
Day 12 1.0e(0.85-1.4)
Day 13 1.0e(0.85-1.4)
Day 14 1.0e(0.85 -1.4)

No Observed Effect Concentration through 14 days: 0.30 mg/L
a LC50 values for exposure days 1, 2 and 3 were empirically estimated as being greater that the highest mean measured test concentration.
bLC50 value for exposure days 4 and 5 were empirically estimated as being equal to the highest mean measured test concentration.
cLC50 value estimated by nonlinear interpolation, lower confidence limit calculated by binomial probability. Upper confidence limit could not be established for this time interval.
dLC50 value and confidence interval calculated by the probit method.
eLC50 value estimated by nonlinear interpolation, confidence interval calculated by binomial probability

Measured concentration of14C-2,6-DTBP in replicate (A,B) solutions during the 14-day flow-through exposure of fathead minnows (Pimephales promelas)

Nominal Conc. (mg/L)

Measured Concentration (mg/L)

Day 0

Day 4

Day 8

Day 12

Day 14

Mean (SD)

A

B

A

B

A

B

A

B

A

B

2.0

1.4

1.5

1.3

1.3

1.4

1.5

1.3

0.89

1.5

1.5

1.4(0.099)

1.3

0.77

0.89

0.75

0.76

0.88

089

0.86

0.60

0.89

0.90

0.85(0.061)

0.84

0.57

0.63

0.48

0.55

0.63

0.61

0.59

0.40

0.69

0.69

0.60(0.064)

0.55

0.39

0.40

0.32

0.34

0.38

0.40

0.40

0.27

0.40

0.41

0.38(0.028)

0.36

0.27

0.27

0.36

0.41

0.28

0.28

0.27

0.28

0.31

0.30(0.047)

Solvent Control

<0.066

<0.066

<0.066

<0.066

<0.066

<0.066

<0.065

<0.065

<0.064

<0.065

Control

<0.066

<0.066

<0.066

<0.066

<0.066

<0.066

0.392a

<0.065

<0.064

<0.064

aMeasured concentration for this replicate indicates the presence of14C-2,6-DTBP in the control solutions. However, it is believed that the sample was contaminated during analytical process and therefore, this measurement is not considered representative of the exposure conditions for this solution.

Concentrations tested and corresponding percent mortalities of fat head minnows (Pimephales promelas) exposed to 2,6 -DTBP during a 14 -day flow-through toxicity test.

Mean Conc. (mg/L)

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Day 7

Mean

Mean

Mean

Mean

Mean

Mean

Mean

1.4

0abc

10afdc

45bd

50bd

55cg

65g

95I

0.85

0h

0abc

0acdf

0abde

0abde

0abd

5ad

0.60

0

0

0

0

0ch

0h

0a

0.38

0

0

0

0

0

0

0

0.30

0

0

0

0

0

0

0

Solvent Control

0

0

0

0

0

0

0

Control

0

0

0

0

0

0

0

 

Mean Conc. (mg/L)

Day 8

Day 9

Day10

Day 11

Day 12

Day 13

Day 14

Mean

Mean

Mean

Mean

Mean

Mean

Mean

1.4

95I

100

100

100

100

100

100

0.85

5ad

5bd

10bd

10bd

10cdh

10cdh

15bd

0.60

0a

0a

0a

0ab

0abcj

0abc

0ab

0.38

0

0

0

0

0c

0c

0h

0.30

0

0

0

0

0

0

0

Solvent Control

0

0

0

0

0

0

0

Control

0

0

0

0

0

0

0

aAll fish at this treatment level were lethargic.

bSeveral fish at this treatment level exhibited partial loss of equilibrium.

cSeveral fish at this treatment level were at the surface of the exposure solution.

dSeveral fish at this treatment level exhibited complete loss of equilibrium.

eOne fish was at the surface of the exposure solution.

fOne fish exhibited partial loss of equilibrium.

gAll surviving fish exhibited complete loss of equilibrium.

hSeveral fish were lethargic.

IThe one surviving fish at this treatment level exhibited a complete loss of equilibrium and was at the surface of the exposure solution.

jSeveral fish at this treatment level had darkened pigmentation.

Conclusions:
The 14 day LC50 was estimated to be 1.0 (0.85-1.4) mg/L 2,6-DTBP for fathead minnows. The comparison of the 7 and 14 day LC50 values, 1.1 and 1.0 mg/L, respectively, suggests that 2,6-DTBP is not chronically toxic to fathead minnows. The No observed Effect Concentration established for the 14-day study was 0.3 mg/L 2,6-DTBP.
Executive summary:

The purpose of this study was to estimate the acute toxicity (LC50) of 2,6-di-tert-Butylphenol (2,6-DTBP) to fathead minnows (Pimephales promelas) under flow-through conditions. In duplicate test aquaria, twenty organisms were exposed to five concentrations of 2,6-DTBP, a dilution water control and a solvent/vehicle (acetone) control, via a flow-through system. Concentrations for this study were based on the results of preliminary range-finding tests conducted at Springborn Life Sciences, Inc. Based on these preliminary data, the following nominal concentrations were selected for the 14-day definitive study: 2.0, 1.3, 0.84, 0.55 and 0.36 mg/L 2,6-DTBP. Concentrations of the test material were maintained in the exposure vessels by introducing approximately 6.3 aquarium volumes per day of newly prepared test solutions via an intermittent-flow proportional diluter apparatus. Each replicate solution was sampled and analyzed for 2,6 -DTBP concentrations prior to test initiation, and on days 0, 4, 8, 12 and 14 of the exposure period. Based on the results of these analyses, the mean measured test concentrations were 1.4, 0.85, 0.60, 0.38 and 0.30 mg/L 2,6-DTBP. Throughout the exposure period a small amount of precipitate was observed in the diluter system’s mixing chamber. However, no undissolved 2,6-DTBP (precipitate, film on solution’s surface) was observed in any of the exposure vessels during the study. Biological observations were made and recorded at test initiation and every 24 hours thereafter until the test was terminated. All fish exposed to the highest test concentration (1.4 mg/L 2,6-DTBP) died within the initial 9 days of the test. At test termination (day 14) 15% mortality was observed at the second highest treatment level (0.85 mg/L) while no mortalities occurred at the remaining treatments (0.60-0.30 mg/L). The 14-day LC50 (95% confidence interval) was estimated by nonlinear interpolation to be 1.0 (0.85-1.4) mg/L 2,6-DTBP. Based on comparison of the 7 and 14 day LC50 values (1.1 and 1.0 mg/L, respectively) 2,6-DTBP does not appear to be chronically toxic to fathead minnows. The No Observed Effect Concentration established for the 14-day study was 0.30 mg/L 2,6-DTBP.

 

Description of key information

LC50 (96 h)  1.4 mg/L

Key value for chemical safety assessment

LC50 for freshwater fish:
1.4 mg/L

Additional information

 

For the endpoint short-term toxicity to fish, four studies are available: Two 96 h-studies and two 14 day studies from which 96h-LC50 data could be obtained.

 

The Klimisch 1 study from Suprenant (1989) with fathead minnow (Pimephales promelas) was performed as 14 -day flow-through study according to OECD 204 and OTS 797.1400. In duplicate test aquaria, twenty organisms were exposed to five concentrations of 2,6-DTBP, a dilution water control and a solvent/vehicle (acetone) control. Nominal concentrations were: 2.0, 1.3, 0.84, 0.55 and 0.36 mg/L 2,6-DTBP. The mean measured test concentrations (mean from day 0 to day 14) were 1.4, 0.85, 0.60, 0.38 and 0.30 mg/L 2,6-DTBP. Biological observations were made and recorded at test initiation and every 24 hours thereafter until the test was terminated. Throughout the 14 day exposure period, no mortality was observed in the control and solvent control. After 96 hours, 50% of the exposed fish had died in the highest test group, i.e. at 1.4 g/L mean measured. In all other treatment groups no mortality occurred within the first 96 hours. Therefore the 96-hour LC50 is 1.4 mg/L. This result is considered to be relevant for the risk assessment.

 

The Klimisch 2 study from Suprenant (1989) with rainbow trout (Oncorhynchus mykiss) was performed as 14 -day flow-through study according to OECD 204 and ASTM E 729 -80. In duplicate test aquaria, twenty organisms were exposed to five concentrations of 2,6-DTBP, a dilution water control and a solvent/vehicle (acetone) control. Nominal concentrations were: 1.5, 0.98, 0.63, 0.41, 0.27 mg/L. 2,6-DTBP. The mean measured test concentrations (mean from day 0 to day 14) were 1.0, 0.66, 0.43, 0.28, 0.21 mg/L 2,6-DTBP. Biological observations were made and recorded at test initiation and every 24 hours thereafter until the test was terminated. Throughout the 14 day exposure period, no mortality was observed in the control and solvent control. After 96 hours, 35% of the exposed fish had died in the highest test group, i.e. at 1.0 mg/L mean measured. In the 0.66, 0.43, 0.28, 0.21 mg/L 2,6-DTBP test groups 15, 10, 65 and 20% mortality was observed within the 96 hours. Therefore the 96-hour LC50 is > 1 mg/L. Due to the weak dose-response relationship, the test is considered to have limited relevance for the risk assessment.

 

Due to experimental shortcomings like lack of dose-verification analysis, the two Klimisch 2 96 hour studies which were preformed according to OECD 203 were considered to be only supporting. In the study from Sewel (1991) the 96h toxicity of 2,6 -DTBP to rainbow trout was determined in a limit test to be > 0.1 mg/L based on nominal concentration (no dose verification analysis). In the study from Rufli (1987) the 96 -hour toxicity of 2,6 -DTBP was determined to be 13 mg/L based on nominal concentration. Since the tested concentrations were above the water solubility limit and no dose verification was performed, this result will not be used for the risk assessment.

 

The acute LC50 of 2,6DTBP to fish is 1.4 mg/L based on the result of the Klimisch 1 study. The 96-day and 14 day Klimisch 2 studies support this result. Therefore, the LC50 of 1.4 mg/L will be used for the risk assessment of 2,6-DTBP.