4-tert-butylpyrocatechol

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 6 June 2013 to 10 October 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

The light intensity and the hardness measurements of the dilution water were below the recommended ranges in the OECD TG 211, but there had no adverse impact on the study results.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

The light intensity and the hardness measurements of the dilution water were below the recommended ranges in the EU method C.20, but there had no adverse impact on the study results.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4-tert-butylpyrocatechol

EC Number:

202-653-9

EC Name:

4-tert-butylpyrocatechol

Cas Number:

98-29-3

Molecular formula:

C10H14O2

IUPAC Name:

4-tert-butylbenzene-1,2-diol

Test material form:

solid: flakes

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were collected from each treatment and control group five days prior to the start of the test after conditioning the diluter for three days. Water samples also were collected from alternating replicate test chambers in each treatment and control group at the beginning of the test, at approximately weekly intervals during the test and at the end of the test to measure concentrations of the test substance. All samples were collected from mid-depth, placed in glass vials, acidified with 3 drops of 10% phosphoric acid (H3PO4) and processed immediately for analysis.

The concentration of the test substance was determined by HPLC method.

Test solutions

Vehicle:

no

Details on test solutions:

Stock solutions were prepared five times during the study. Based upon the information provided by the sponsor, it appears that the test substance is more stable at lower pH. Therefore, the stock solutions were prepared in acidified reversed osmosis (RO) water to ensure stability. A primary stock solution was prepared by mixing a calculated amount of test substance into acidified RO water at a nominal concentration of 3965 µg/mL and mixed by sonication for approximately 10 minutes followed by inversion at least 20 times. Four secondary stock solutions were prepared in RO water at nominal concentrations of 99.4, 258, 636 and 1590 µg/mL by proportional dilution of the primary stock. The secondary stock solutions were mixed by inversion at least 20 times, and all appeared clear and colorless with no visible precipitate noted. Stock solutions were stored refrigerated in glass amber bottles, and aliquots of each stock were placed in the syringe pump every two days during the study.

The five test substance stock solutions were injected into the diluter mixing chambers at a rate of 15.6 µL/minute where they were mixed with dilution water delivered at a rate of 155 mL/minute to achieve the desired test concentrations. The negative control received dilution water only.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
Daphnids are representative of an important group of aquatic invertebrates and were selected for use in the test based upon past history of use in the laboratory.
- Source: Wildlife International, Easton, Maryland.
- Age of parental stock: less than 24 hours old
- Feeding during test: yes
- Food type: a mixture of yeast, cereal grass media, and trout chow (YCT), supplemented with a vitamin stock solution and a suspension of the freshwater green alga, Pseudokirchneriella subcapitata.
- Amount: approximately 0.6 to 0.7 mg C/daphnid/day: At each feeding on Days 0 to 12 of the test, each test chamber was fed 0.6 mL of YCT, 1.2 mL of algae and 0.40 mL of vitamin solution. At each feeding on Days 14 to 21, each test chamber was fed 0.75 mL of YCT, 1.5 mL of algae and 0.50 mL of vitamin solution. On Day 13 of the test, each test chamber was fed 0.6 mL of YCT, 1.2 mL of algae and 0.40 mL of vitamin solution during the first feeding and was fed 0.75 mL of YCT, 1.5 mL of algae and 0.50 mL of vitamin solution during the three additional feeding on that day. This amount of feed is equal to approximately 0.6 to 0.7 mg C/daphnid/day. While this amount of feed exceeds the OECD guideline recommended amount of 0.1 to 0.2 mg C/daphnid/day, an excess amount was fed in order to maintain sufficient feed in the flow-through system to support acceptable reproduction rates.
- Frequency: Daphnids were fed three to four times per day until Day 20 of the test and once during the last day of the test.

ACCLIMATION
- Acclimation period: Adult daphnids were cultured in water from the same source and at approximately the same temperature as used during the test. During the 2 week period immediately preceding the test, water temperatures in the cultures ranged from 19.5 to 20.8ºC, measured with a hand-held, liquid-in-glass thermometer. The pH of the water ranged from 8.2 to 8.5, measured with a Thermo Orion Model 525Aplus pH meter. Dissolved oxygen concentrations were ≥ 7.6 mg/L (≥ 84% of saturation), measured with a Thermo Orion Model 850Aplus dissolved oxygen meter.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS
The four adult daphnids used to supply neonates for the test were held for at least 13 days prior to collection of the juveniles for testing, and had each produced at least one previous brood. Adult daphnids in the culture had produced an average of at least three young per adult per day over the 7 day period prior to the test. The adults showed no signs of disease or stress and no ephippia were produced during the holding period. To initiate the test, the juvenile daphnids were collected from the cultures and indiscriminately transferred one or two at a time to transfer chambers until each chamber contained 5 daphnids. Each group of neonates then was impartially assigned to a control or treatment group and the neonates were transferred to the test compartments to initiate the test. All transfers were made below the water surface using wide-bore pipettes.

Study design

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Test conditions

Hardness:

Hardness was above 134 mg/L (as CaCO3). The hardness measurements of the dilution water during the test were slightly below the recommended 140 mg CaCO3/L, but were well within the typical ranges of Wildlife International well water.

Test temperature:

The tempareture remained constant within a range from 19.9°C to 20.1°C during the study. The target test temperature during the test was 20 ± 1°C. Temperature was measured in one replicate test chamber of each treatment or control group at the beginning of the test, and in each test chamber approximately weekly during the test, and at the end of the test using a liquid-in-glass thermometer. Temperature also was monitored continuously in one negative control test chamber using a Fulscope ER/C recorder. The recorder was verified prior to test initiation and approximately weekly during the test using a liquid-in-glass thermometer.

pH:

The pH remained constant at 8.1 during the study. Measurements of pH were made in one replicate test chamber of each treatment and control group at the beginning of the test, approximately weekly during the test, and at the end of the test.

Dissolved oxygen:

Between 8.3 and 8.4 mg/L during the study. Dissolved oxygen was measured in one replicate test chamber of each treatment and control group at the beginning of the test, approximately three times per week during the test, and at the end of the test.

Nominal and measured concentrations:

Nominal concentrations: 0, 10, 26, 64, 160 and 400 µg/L
Mean measured concentrations: 0, 6.3, 19, 56, 135, 359 µg/L
The results of the study were based on the mean measured concentrations.

Details on test conditions:

TEST SYSTEM
Based on the information provided by the sponsor, the substance was found to be unstable in a GLP Daphnia magna 48-hour immobilization study performed in semi-static conditions. Additionally, the substance was found to be stable at pH 4, but not stable at pH 7 and 9 in an hydrolysis study. Therefore, the study was conducted using an exposure system consisting of a continuous-flow diluter used to deliver each concentration of the test substance and a negative control (dilution water) to test chambers. Syringe pumps (Harvard Apparatus, South Natick, Massachusetts) were used to deliver test substance stock solutions to impartially assigned mixing chambers where the stocks were mixed with dilution water prior to delivery to the test chambers. The flow of dilution water into each mixing chamber was controlled using rotameters and was adjusted to provide approximately five volume additions of test water in each test chamber per day. After mixing, the flow from each mixing chamber was split to deliver test water to two replicate test chambers.
The syringe pumps used to deliver stock solutions to the mixing chambers were calibrated prior to the test. The rotameters used to control the flow of dilution water to the mixing chambers were calibrated prior to the test and calibrated/verified approximately weekly during the test. The proportion of the test water that was split into each replicate test chamber was checked prior to the test and approximately weekly during the test to ensure that flow rates varied by no more than ± 10% of the mean flow rate for the two replicates. Delivery of test solutions to the test chambers was initiated eight days prior to the introduction of the test organisms to the test water in order to achieve equilibrium of the test substance. The general operation of the exposure system was checked visually at least once on the first and last days of the test and at least two times per day during the test.

- Test vessel: Test chambers were 25 L Teflon®-lined stainless steel aquaria filled with approximately 22 L of test water. The daphnids were held in two test compartments suspended in each of two test chambers. Test compartments were 300 mL glass beakers, approximately 6.5 cm in diameter and 12 cm in height. Nylon mesh screens covered two holes on opposite sides of each test compartment to permit test solution to flow in and out of the compartment. The depth of the test water in a representative compartment was approximately 8 cm, while the depth of water in a representative test chamber was approximately 29 cm. All test chambers were labeled with the project number, test concentration and replicate designation.
- Type: closed
- Aeration: no
- Type of flow-through: continuous-flow diluter
- No. of organisms per vessel: Each replicate contained two compartments with five daphnids.
- No. of vessels per concentration (replicates): 2, resulting in a total of 20 daphnids in each treatment
- No. of vessels per control (replicates): 2, esulting in a total of 20 daphnids in control group.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water used for culturing and testing was freshwater obtained from a well approximately 40 meters deep located on the Wildlife International site. The well water was passed through a sand filter to remove particles greater than approximately 25 µm, and pumped into a 37,800 L storage tank where the water was aerated with spray nozzles. Prior to use, the water was filtered to 0.45 µm to remove fine particles and was passed through an ultraviolet (UV) sterilizer. The well water is characterized as moderately-hard water.
- Total organic carbon: < 1 mg C/L
- Specif conductance: 359 (342-373) µS/cm
- Hardness: 140 (136-144) mg/L as CaCO3
- Alkalinity: 178 (176-180) mg/L as CaCO3
- pH: 8 (8 - 8.1)
- Culture medium different from test medium: no
- Intervals of water quality measurement: TOC is measured monthly. The above parameters were measured during the 4-week period immediatly preceding the test.

OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Photoperiod: 16 hours of light / 8 hours of darkness, daily
- Light intensity: 378 lux. Since the study met all of the conditions for the validity of the test, it is apparent that the lower light intensity used in the study had no adverse impact on the study results.

EFFECT PARAMETERS MEASURED: Observations of each parent animal were made daily during the test. At these times, the numbers of immobile daphnids were recorded along with any clinical signs of toxicity (e.g., inability to maintain position in the water column, uncoordinated swimming or cessation of feeding). Immobility was defined as a lack of movement, except for minor spontaneous random movement of the appendages. The presence of eggs in the brood pouch, aborted eggs, males or ephippia also was recorded daily. With the onset of reproduction, neonates produced by the parent animals were counted and then discarded during the test (e.g. every Monday, Wednesday and Friday). The body length and the dry weight of each surviving parent animal were measured at the end of the test.

RANGE-FINDING STUDY
- Test concentrations: 0, 7.3, 24, 81, 270 and 900 µg/L
A 14-day preliminary range finding test was conducted to determine the approximate toxicity of the test item on the reproductive output, so that appropriate test concentrations can be selected for use in the definitive test. 10 test animals were exposed in every concentration level and also in the control. Results of the preliminary range-finding test is presented in Table 1.
- Results used to determine the conditions for the definitive study: In the definitive test 5 test item concentrations were investigated. The concentrations were chosen based on the preliminary concentration-range finding test. An untreated control group and the following concentrations were tested in the study: 0, 10, 26, 64, 160 and 400 µg/L.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 359 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Effect concentrations 2

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 359 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 3

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

135 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 4

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

359 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- MEASUREMENT OF TEST CONCENTRATIONS
The nominal concentrations were tested: 10, 26, 64, 160 and 400 µg/L.
During the course of the test, the appearance of the test solutions at these nominal concentrations was observed in both the diluter mixing chambers, where test substance stocks and dilution water were combined prior to delivery to the test chambers, and in the test chambers. The test solutions in the mixing chambers and test chambers appeared clear and colorless during the test, with no evidence of precipitation observed in any control or treatment solution.
Samples of the test solutions collected during the test had measured concentrations that ranged from 55.4 to 97.0% of nominal concentrations. The measured concentrations of the samples collected from the 10 and 26 µg/L nominal test concentrations had recoveries below 80% of nominal. However, the measured concentrations were consistent throughout the study (with the %CV of 9.3 and 3.9, respectively). When the measured concentrations of test solution samples collected on Days 0, 7, 14 and 21 of the test were averaged for each treatment group, the mean measured test concentrations were 6.3, 19, 56, 135 and 359 µg/L representing 63, 73, 88, 84 and 90% of nominal concentrations, respectively. The results of the study were based on the mean measured concentrations.

- SURVIVAL AND CLINICAL OBSERVATIONS
A summary of survival among the parent animals is presented in Table 2. After 21 days of exposure, survival in the negative control group was 95%. Survival in the 6.3, 19, 56, 135 and 359 µg/L treatment groups at test termination was 100, 90, 95, 95 and 95%, respectively. Fisher’s Exact test indicated that there were no statistically significant decreases in survival in any of the 4-TBC treatment groups in comparison to the negative control (p > 0.05).
Daphnids in the 6.3, 19, 56, 135 and 359 µg/L treatment groups that survived to test termination generally appeared normal.

- REPRODUCTION
A summary of reproduction based on the total number of living neonates produced per adult daphnid present at the beginning of the test and as the number of live neonates produced per reproductive day are presented in Table 2.
The first day of brood production in the negative control replicates and in all 4-TBC treatment replicates was Day 8 or 9 of the test, indicating that there was no apparent delay in the onset of production at any 4-TBC concentration tested. No immobile neonates were noted in the control group or the 6.3 and 56 µg/L treatment groups during the test, while six, one and seven immobile neonates noted in the 19, 135 and 359 µg/L treatment groups, respectively. The occurrence of immobile neonates was not dose-responsive and limited in number, and was not considered to be treatment related. No aborted or shed eggs were noted in the control group or the 6.3, 19 and 56 µg/L treatment groups during the test. At the end of the study, the total numbers of aborted or shed eggs observed in the 135 and 359 µg/L treatment concentrations were 23 and 128, respectively. Observation of the aborted or shed eggs in the 135 µg/L treatment group was limited to the first day of brood production on Day 8 of the test. No aborted or shed eggs were observed in the 135 µg/L treatment group after the first day of brood production and aborted or shed eggs observed on Day 8 appeared to be incidental to treatment. Aborted eggs in the 359 µg/L treatment concentration were noted on Days 8, 10, 15, 17 and 21 of the test. No males or ephippia were produced during the test.

Dunnett’s test indicated there was a statistically significant decrease in reproduction based on both the mean number of live neonates per adult present at test initiation and on the mean number of live young per reproductive day in the 359 µg/L treatment group in comparison to the negative control (p ≤ 0.05). Although there were no statistically significant differences noted in the total number of live neonates produced per surviving adult at test end in any of the 4-TBC treatment groups when compared to the negative control (Dunnett’s one-tailed test, p > 0.05), an apparent decrease could be seen at the 359 ug/L treatment level. The reason that Dunnett’s did not detect the statistically significant decrease in this treatment level is due to the wide standard deviations noted throughout.

The 21-day EC50 value for the reproduction observed in the treatment groups, based on both the mean number of live neonates per adult present at test initiation and on the mean number of live young per reproductive day, was greater than 359 μg/L, the highest concentration tested. The 21-day EC10 value, based on the mean number of live neonates per adult present at test initiation, was 207 μg/L, with the 95% confidence interval of 4.4 to 296 μg/L. The 21-day EC10 value, based on the mean number of live young per reproductive day, was 169 μg/L with the 95% confidence interval of -47.8 to 281 μg/L

- GROWTH
Summaries of the mean lengths and dry weights of surviving parent animals is presented in Table 2. Dunnett’s test indicated there was a statistically significant decrease in mean dry weight in the 135 µg/L treatment group in comparison to the negative control (p ≤ 0.05) but no statistically significant decreases in mean total length in any of the 4-TBC treatment groups in comparison to the negative control (p > 0.05). Since the mean dry weight in the 135 µg/L treatment group did not follow a dose response pattern, the statistically significant difference found in this treatment group was not considered to be biologically meaningful or treatment related.

Reported statistics and error estimates:

Test endpoints analyzed statistically for parent animals were survival, reproduction, and growth (length and dry weight). Reproductive endpoints were expressed as the total number of living neonates produced per adult daphnid present at the beginning of the test, as the total number of living neonates produced per surviving adult daphnid at test termination and as the number of live neonates produced per reproductive day. Reproductive days were defined as the number of days that the adult daphnid was alive from the day the first brood was released from any adult daphnid in the test until test termination. If an adult daphnid died, the number of reproductive days, for that adult, ended on the last day it was alive.
Survival data were analyzed using Fisher’s Exact test to identify treatment groups that showed a statistically significant difference (α = 0.05) from the control.
Reproduction and growth data were evaluated for normality using Shapiro Wilk’s or Kolmogorov-Smirnov test and for homogeneity of variance using Levene’s test (α = 0.01). When the data passed the assumptions of normality and homogeneity, those treatments that were significantly different from the control means were identified using Dunnett’s test (α = 0.05).
All statistical tests were performed using a personal computer with TOXSTAT (version 3.5) or SAS (version 8.2) software.

Any other information on results incl. tables

Table 2: Summary of survival, reproduction and growth of Daphnia magna exposed to 4 -TBC for 21 days

Mean Measured Concentration (µg/L)	Percent Adult Survival^1,2	Mean No. Neonates Per Reproductive Day ± Std. Dev.^3	Mean No. Neonates Per Adult Present at Test Initiation ± Std. Dev.	Mean No. Neonates Per Surviving Adult at Test Termination ± Std. Dev.^1	Mean Length ± Std. Dev.^1 (mm)	Mean Dry Weight ± Std. Dev. (mg)
Negative Control	95	12.2 ± 0.68	160.8 ± 12.8	169.8 ± 9.6	4.8 ± 0.05	1.45 ± 0.15
6.3	100	11.0 ± 1.27	154.5 ± 18.1	154.5 ± 18.1	4.7 ± 0.05	1.34 ± 0.16
19	90	12.7 ± 0.57	173.7 ± 10.0	201.1 ± 49.0	4.8 ± 0.08	1.46 ± 0.03
56	95	11.1 ± 0.50	155.0 ± 7.5	164.3 ± 16.1	4.7 ± 0.10	1.38 ± 0.13
135	95	11.9 ± 2.15	165.7 ± 29.1	178.5 ± 53.3	4.7 ± 0.16	1.19 ± 0.21*^,4
359	95	9.4 ± 0.50*	127.9 ± 11.6*	134.9 ± 7.1	4.7 ± 0.05	1.33 ± 0.10
*  Indicates a statistically significant decrease in comparison to the negative control (p ≤ 0.05). ^1  There were no statistically significant decreases in survival (Fisher’s Exact test, p > 0.05), mean number of neonates produced per surviving adult at test end, or mean total length in comparison to the negative control (Dunnett’s one-tailed test, p > 0.05). ^2  21-day EC50 for survival: >359 µg/L. ^3  21-day EC50 for reproduction: >359 µg/L. ^4Since the mean dry weight of the 135 µg/L treatment concentration did not follow a dose-response pattern, the statistically significant difference found in this treatment level was not considered to be biologically meaningful or treatment related.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

(Immobility of the parents in the control(s) or accidental and inadvertant parental immobility did not exceed 20% at the end of the test. The mean number of live offspring per parent animal surviving at the end of the test was > 60.)

Conclusions:

The cladoceran, Daphnia magna, was exposed to 4-TBC at mean measured concentrations of 6.3 to 359 µg/L under flow-through conditions for 21 days. There were no significant treatment-related effects on survival or growth at concentrations ≤359 µg/L. Reproduction, expressed as the total number of live neonates produced per parent animal present at test initiation or as number of live neonates produced per reproductive day, was the most sensitive biological endpoint measured in this study. Daphnids exposed to 4-TBC at concentrations ≥359 µg/L had statistically significant reductions in reproduction in comparison to the negative control. Consequently, the NOEC, based on reproduction, was 135 µg/L. The LOEC was 359 µg/L.

The 21-day EC50 values for immobility of parent animals and for reproduction were both greater than 359 µg/L, the highest concentration tested. The 21-day EC10 value for immobility of parent animals also was greater than 359 µg/L. The 21-day EC10 value, based on the mean number of live neonates per adult present at test initiation, was 207 µg/L, with the 95% confidence interval of 4.4 to 296 µg/L. The 21-day EC10 value, based on the mean number of live young per reproductive day, was 169 µg/L with the 95% confidence interval of -47.8 to 281 µg/L.

Executive summary:

The objective of the study (2013) was to determine the effects of 4 -tert-butylpyrocatechol (4 -TBC) on the survival, growth and reproduction of the cladoceran,Daphnia magna, during a 21 -day exposure period under flow-through test conditions.

The study was performed according to the OECD 211 guideline and under GLP conditions.

Daphnids were exposed to a geometric series of five test concentrations and a negative control (dilution water). Two replicate test chambers were tested for each treatment and control group. Each replicate contained two compartments with five daphnids, resulting in a total of 20 daphnids in each treatment and control group. The nominal test concentrations were 10, 26, 64, 160 and 400 µg/L. Mean measured test concentrations were determined from samples of test water collected from each treatment and control group at test initiation, at approximately weekly intervals during the test and at test termination.

As the measured concentrations deviated more than 20 per cent from the nominal in some cases, biological results are based on the mean measured concentrations. The corresponding mean measured concentrations were: 6.3, 19, 56, 135 and 359 µg/L.

 

Parent animals were observed daily during the test for mortality, the onset of reproduction, and clinical signs of toxicity. Following the onset of reproduction, the numbers of offspring were counted three times per week, and at test termination (Day 21). Body lengths and dry weights of the surviving parent animals were measured at the end of the exposure period. Observations of the effects of 4-TBC on survival, reproduction and growth were used to determine the no-observed-effect concentration (NOEC), the lowest-observed-effect concentration (LOEC). EC50 and EC10 values were determined based on reproduction and on parent animals immobility at test termination.

There were no significant treatment-related effects on survival or growth at concentrations ≤ 359 µg/L. Reproduction, expressed as the total number of live neonates produced per parent animal present at test initiation or as number of live neonates produced per reproductive day, was the most sensitive biological endpoint measured in this study. Daphnids exposed to 4-TBC at concentrations ≥ 359 µg/L had statistically significant reductions in reproduction in comparison to the negative control. Consequently, the NOEC, based on reproduction, was 135 µg/L and the LOEC was 359 µg/L.

The 21-day EC50 values for immobility of parent animals and for reproduction were both greater than 359 µg/L, the highest concentration tested. The 21-day EC10 value for immobility of parent animals also was greater than 359 µg/L. The 21-day EC10 value, based on the mean number of live neonates per adult present at test initiation, was 207 µg/L, with the 95% confidence interval of 4.4 to 296 µg/L. The 21-day EC10 value, based on the mean number of live young per reproductive day, was 169 µg/L with the 95% confidence interval of -47.8 to 281 µg/L.

The biological results were summarized as follows (based on measured concentrations):.

- 21-day EC50, Immobility:	> 359 µg/L
- 21-day EC50, Reproduction:	> 359 µg/L
- 21-day NOEC, Reproduction:	135 µg/L
- 21-day LOEC, Reproduction:	359 µg/L

Based on the results, 4 -TBC was toxic to daphnids with long lasting effects.

This study is classified acceptable and satisfies the guideline requirements for chronic daphnids toxicity study.