N-tert-butylbenzothiazole-2-sulphenamide

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:

1997-01-16 - 1997-05-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

also cited in OECD SIDS.

Qualifier:

according to guideline

Guideline:

other: OECD Guide-line 202, part 2 "Daphnia sp., Reproduction Test"

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Purity 96.8 %, Batch No. 612040

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
The test materials were stored in a refrigerator in our laboratory.
The infrared absorption spectra of the available test substance were measured and confirmed to be consistent with the structure of the test substance. At the end of the test, the infrared absorption spectrum was also measured and compared with the spectrum measured before the start of the test. As a result, there was no change in the spectrum, and the test substance was judged to have been stable during storage

Analytical monitoring:

yes

Details on sampling:

For all concentration groups (one test vessel for each), 0.75 mL of each test solution at the beginning of exposure and before water change was collected in a measurement vial and analyzed by HPLC after adding an equal volume of acetonitrile. The standard solutions prepared with acetonitrile were diluted with an equal volume of water and used as HPLC measurement samples. The concentration of the test substance in each test solution was determined from the ratio of the peak area to that of the standard solution.

Vehicle:

yes

Remarks:

Hardened castor oil HCO-50 and DMF

Details on test solutions:

STOCK AND TEST SOLUTION AND THEIR PREPARATION:
Vehicle, solvent: HCO-50 and DMF
Concentration of vehicle/solvent: 30 mg/L

Preparation of the test solution
200 mg of the test substance was weighed and dissolved in 400 mg of DMF, and further stirred and mixed after the addition of 19600 mg of HCO-50. This solution was diluted with pure water to a constant volume of 1000 mg/L, and a stock solution with a test substance concentration of 200 mg/L was prepared. At the same time,
a solvent stock solution of 2000 mg/L (DMF 400 mg/L, HCO-50 19600 mg/L) without the test substance was prepared.
Add 800 mL of diluted water in a 1.0 L glass beaker, add the above test substance solution according to each concentration, add the solvent stock solution so that the solvent concentration becomes constant (30 mg/L), and perform each test. The liquid was prepared. Solvent control group containing no test substance (solvent concentration: 30 mg/L) were prepared. Only diluted water was used in the control group.


STABILITY OF THE TEST CHEMICAL SOLUTIONS:
The concentration of TBBS in the test solution at the time of preparation of the test solution was 0.019 - 0.304 mg/L (set-up value 0.02 - 0.3 mg/L) and the concentration before the exchange of solution was 0.004 - 0.021 mg/L. In addition the ratios of test material concentration measured to the set-up value in each concentration range were 88 - 135 % at the time of preparation and 5 - 25 % before the exchange.
The concentration of TBBS measured at preparation and before exchange during the exposure period were +/- 20 % of the set-up values, therefore, the measured (time-weighted average) were used.

Dilution water
Dechlorinated water (Yokohama city tap water treated with activated carbon to remove residual chlorine, etc., and then thoroughly aerated) was used. The hardness of the diluted water was 65 mg/L (CaCO3 count) and the pH was 8.2.

Test organisms (species):

Daphnia magna

Details on test organisms:

Juvenile Daphnia magna (Daphnia magna) within 24 hours of birth were used in this study.
The juveniles of Daphnia magna were obtained from the National Institute for Environmental Studies (NIES) on July 18, 1995 and have been reared in our laboratory in succession. The concentration of the substrate (potassium dichromate, reagent grade) inhibiting half swimming (EiC50) was 0.23 mg/L in 48 hours.

Daphnia breeding method for obtaining juveniles to be tested.

The juveniles were then transferred to a separate beaker, and the next day, the produced juveniles were placed in a separate beaker. Daphnia magna juveniles (born on the 16th day of April, 1997) were used as test specimens and reared under the following conditions. The juveniles were removed twice a week when they matured and began to produce juveniles. The juveniles were removed twice a week when they reached maturity and began to produce juveniles. At the beginning of the third week, on the day before the start of the exposure, adult females with juveniles in their eggs were selected from buffleheads that had 0% mortality and no dormant eggs or males during the previous two weeks, and juveniles produced the next day (within 24 h) were used for the test (exposure started on May 6, 1997).

1) Breeding water : Dilution water
2) Breeding density : As juveniles: 35 or 70 animals / 3 L breeding water
As adults: 35 animals´/ 3 L breeding water
3) Temperature : 20+1 °C
4) Illumination : Room light, 16 hours light / 8 hours dark
5) Feeding : Chlorella vulgaris
6) Feeding amount : 0.1～0.2 mgC (organic carbon content) per Daphnia / day

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

The hardness of the diluted water was 65 mg/L (CaCO3 equivalents).

Test temperature:

19.8 - 20.3 °C

pH:

7.3 - 8.1

Dissolved oxygen:

6.7 - 8.5 mg/L

Nominal and measured concentrations:

nominal concentrations: 0.020, 0.040, 0.080, 0.15 and 0.3 mg/L plus control and vehicle control
time-weighted mean during 21 days: 0.012, 0.022, 0.042, 0.068, 0.16 mg/L

Details on test conditions:

Test conditions

1) Exposure method : Semi-static (replace the entire amount of test solution after 48 hours)
2) Exposure period : 21 days
3) Test water volume : 800 mL
4) Number of stations : 4 vessels per 1 concentration group
5) Number of organisms : 40 neonates/1 concentration group (40 neonates for 1 concentration group,
10 neonates per 1 vessel)
6) Test water temperature : 20+1 °C
7) Illumination : Room light, 16 hours light / 8 hours dark
8) Feeding : Chlorella vulgaris
9) Feeding amount : 0.1～0.2 mgC (organic carbon content) per Daphnia / day

Dilution water

Dechlorinated water (Yokohama city tap water treated with activated carbon to remove residual chlorine, etc., and then thoroughly aerated) was used. The hardness of the diluted water was 65 mg/L (CaCO3 count) and the pH was 8.2.

Test container, constant temperature bath, etc.

1) Test vessel : 1L glass beaker
2) Thermostatic bath : PVC tank (thermostat TAITEC COOLNIT CL-80F type)
3) Water thermometer : Yokogawa Denki 2455 02
4) Dissolved oxygen meter : Electrochemical measuring instrument DOL-10
5) pH meter : HM-40V model made by Toa Denpa Kogyo

Test concentration design
The results of the 48-hour acute swimming inhibition test for Daphnia magna showed that the half swimming inhibition concentration (48hr-EiC50 value) was 1.31 mg/L (using 100 mg/L of solvent). In the previous study on the dispersion method, it was confirmed that the higher the amount of solvent relative to the amount of test substance, the more stable the test substance concentration was. In this study, we examined the preparation method of the test solution in consideration of the concentration of the solvent that would not affect the breeding of Daphnia magna. As a result, in this study, the maximum concentration was set at 0.3 mg/L (the upper limit concentration that can be prepared), and five levels were set at a common ratio of 2.0. Each concentration group is as follows.

Control, Solvent control, 0.020, 0.040, 0.080, 0.150, 0.300 mg/L

Test method
After measuring the temperature of the test solution, the residual oxygen concentration (DO), and the pH, the test Daphnia was added, and the time point was nominal as the start of exposure. The test Daphnia was put in using a pipettor with a thick tip. At that time, the total amount of breeding water in the pipette should be within 1% of the test water volume. After that, Daphnia were transferred to a new test solution after each water change and bred until the 21st day. During the exposure period, a fixed amount of Daphnia was fed every day.

Observation of daphnia:
(Parent Daphnia) The number of surviving Daphnia magna, the number of inhibited swimmers, the number of individuals with eggs in the growth cells, and their size and condition were compared with those of the control and recorded daily. After counting, dead parental Daphnia magna were removed.

(First brood): after larval production, the juveniles were counted alive or dead. The occurrence of aborted and dormant eggs was observed and recorded at the time of water change. The juveniles were removed after counting. The date of the first larval production was recorded as the first production.

Water quality measurement:
Water temperature, dissolved oxygen concentration (D.O.), and pH were measured twice during the exposure period (once at the time of preparation and once before water change two days later) for all concentration groups (one test vessel for each) before and after water change. The measurements were made twice during the exposure period (once at the time of preparation and once two days later before water change).

Reference substance (positive control):

yes

Remarks:

The concentration of the substrate (potassium dichromate, reagent grade) inhibiting half swimming (EiC50) was 0.23 mg/L in 48 hours.

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

> 0.16 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 0.16 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

> 0.16 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.068 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: parental

Details on results:

In the 0.150 and 0.300 mg/L (measured values: 0.068 and 0.160 mg/L) groups, the increase in the number of juveniles was suppressed compared to the control and solvent control groups up to 14 days after the start of exposure, but after 14 days, the increase in the number of juveniles exceeded that in the other concentration groups, and the cumulative number of juveniles exceeded that in the control group. However, after 14 days, the number of juveniles increased more than other concentration groups, and the cumulative number of juveniles exceeded that of the control group and almost recovered to the value of the solvent control group.
Therefore, the test substance may affect Daphnia magna in the juvenile stage, although recovery was observed after 21 days of exposure to 0.068 mg/L or more.

Results with reference substance (positive control):

The concentration of the substrate (potassium dichromate, reagent grade) inhibiting half swimming (EiC50) was 0.23 mg/L in 48 hours.

Reported statistics and error estimates:

Calculation of half lethal concentration (LC50) of parent Daphnia magna
The mortality rate was calculated from the number of dead Daphnia magna in each concentration group and the number of test animals (40), and the half lethal concentration (EiC50) was calculated using the Probit method.

ErC50 calculation:
The inhibition ratio of cumulative juveniles in each concentration group to cumulative juveniles in the solvent control group was determined, and the 50% breeding inhibition concentration (ErC50) was calculated by Probit using the value. However, the inhibition ratio was not found in the highest concentration group as it was 50% or more.

No observed effect concentration (NOECr) and Lowest observed effect concentration (LOECr)
The cumulative number of juveniles per parent of each test container is calculated, and the presence or absence of a significant difference between each concentration and an solvent control group is determined by multiple comparison tests (α=0.05, both sides) of Dunnett. The maximum concentration (no observed effective concentration: NOECr) at which no significant difference is found and the minimum concentration
(lowest observed effect concentration: LOECr) at which a significant difference is found are determined.
For statistical analysis, Yukms software Statlight"#4 multigroup comparison" (Yukms Corp., Tokyo) was used.

Number of mortalities and death rate:

Death rates of parent Daphnia in the control and vehicle control were 15 % and 7.5 % respectively by the end of the exposure period. These values fulfilled the standard of less than 20 % which is the requirement of the study.

In the test groups (based on measured concntrations) the death rates were as follows:

0.012 mg/L = 5 %

0.022 mg/L = 2.5 %

0.042 mg/L = 5 %

0.068 mg/L = 5 %

0.16 mg/L = 35 %

Day of first birth: In the control and vehicle control the first day of birth was 7 days after the start of exposure and fulfilled the standard level within 9 days which is the requirement for the study. In the test groups the day of first birth was 7 days for the 0.012, 0.022, 0.042 and 0.068 mg/L dose levels (based on measured test concentrations) and 10 days for the 0.16 dose level.

Mean value of accumulated offspring (based on measured concentrations):

Control group = 82

Vehicle control = 96

0.012 mg/L = 103

0.022 mg/L = 105

0.042 mg/L = 108

0.068 mg/L = 87

0.16 mg/L = 88

Lowest observed effect concentration (LOECr) on accumulated number of born offspring per one parent Daphnia > 0.16 mg/L (measured).

Size and condition of parent: There were no observable differences in the size of parent Daphnia between the control, the vehicle control and the 0.012 and 0.022 mg/L test groups. In comparison to the control group the parent Daphia was small on day 9 in the 0.042 mg/L group, on days 7-15 in the 0.068 mg/L group and on days 7-17 in the 0.16 mg/L group.

Dormant eggs: There were no dormant eggs in any of the groups throughout the study.

Numbers of offspring: Numbers of offspring in the 0.068 and 0.16 mg/L dose groups were inclined to decrease compared with those in the control and vehicle control groups until 14 days after exposure. However, numbers of offspring increased after 14 days in comparison with the other test groups. The numbers exceeded those in the control group and recovered to the level of those in the vehicle control group.

Validity criteria fulfilled:

not applicable

Conclusions:

It was considered that the test material might affect the larva of Daphnia by exposure of 0.068 mg/L or more after 14 days, although recovery in reproduction was observed and there is no significant difference between the 0.160 mg/L and control at 21 days of exposure (end of the test period)

Executive summary:

In this study the toxicity of N-tert-butlybenzothiazole-2-sulphenamide to Daphnia magna was evaluated according to OECD 202 part II, albeit with a test duration of 21 days. Juvenile Daphnia magna (Daphnia magna) within 24 hours of birth were used in this study. After measuring the temperature of the test solution, the residual oxygen concentration (DO), and the pH, the test Daphnia was added, and the time point was nominal as the start of exposure. The test Daphnia was put in using a pipettor with a thick tip. At that time, the total amount of breeding water in the pipette should be within 1% of the test water volume. After that, Daphnia were transferred to a new test solution after each water change and bred until the 21st day. During the exposure period, a fixed amount of Daphnia was fed every day.

Following a preliminary acute toxicity test the nominal concentrations were set to be 0.020, 0.040, 0.080, 0.15 and 0.3 mg/L plus control and vehicle control. Since the concentrations of the test substance measured during the exposure period exceeded ±20% of the nominal values, the measured values (time-weighted averages) were used to calculate the effect concentrations.

time-weighted mean during 21 days: 0.012, 0.022, 0.042, 0.068, 0.16 mg/L

 

The determined 21d-E_rC₅₀, LC₅₀, NOEC_r and LOEC_r are all >0.16 mg/L.

 

However, in the 0.150 and 0.300 mg/L (measured values: 0.068 and 0.160 mg/L) groups, the increase in the number of juveniles was suppressed compared to the control and solvent control groups up to 14 days after the start of exposure. After 14 days, the increase in the number of juveniles exceeded that in the other concentration groups, and the cumulative number of juveniles exceeded that in the control group. However, after 14 days, the number of juveniles increased more than other concentration groups, and the cumulative number of juveniles exceeded that of the control group and almost recovered to the value of the solvent control group.
Therefore, the test substance may affect Daphnia magna in the juvenile stage, although recovery was observed after 21 days of exposure to 0.068 mg/L or more.

Description of key information

Regarding the reproduction rate for TBBS, the 21day-NOEC for Daphnia magna was > 0.16 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.16 mg/L

Additional information

Should read ">0.16 mg/L"

Due to fast hydrolysis of TBBS the observed effect is most probably a combined effect from both, TBBS and MBT. Thus, the value determined on TBBS was chosen as key study. However the effective concentration on invertebrates reported for MBT is less sensitive than the one reported for TBBS on algae, and hence this value was not used in further risk assessment for the PNEC estimation.