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Long-term toxicity to fish

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fish, juvenile growth test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 Aug - 12 Nov 2013
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to
OECD Guideline 215 (Fish, Juvenile Growth Test)
GLP compliance:
Analytical monitoring:
Details on sampling:
- Concentrations: the control and the treatment with 100 mg/L (nominal) test item were sampled
- Sampling method: 2 mL test solution was taken from each test vessel at the beginning, before and after each renewal and at the end of the test
- Sample storage conditions before analysis: samples were stored in refrigerator (5 ± 3 °C) when not analyzed directly
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: a nominal concentration of 100 mg/L of the test substance was prepared and stirred in the dark (72 h, 500 rpm). Solution was then filtrated through a 0.451 µm nitrocellulose filter to make a saturated solution in the test water
Test organisms (species):
other: Chinese Rare Minnow (Gobiocypris rarus)
Details on test organisms:
- Common name: Chinese Rare Minnow
- Source: fish were bred in Bioassay and Safety Assessment Laboratory, Shanghai Academy of Public Measurement
- Age at study initiation: juvenile fish in exponential growth phase
- Weight at study initiation (mean and range, SD): 0.0765 g (control), 0.0757 g (treatment group)
- Feeding during test: yes
- Amount: 4% of body weight / d
- Frequency: twice a day, two feeds were separated by 6 h

- Acclimation period: 14 d
- Acclimation conditions: tap water dechlorinated by activated carbon and sterilized by UV lamp was used; temperature: 21 °C - 25 °C; pH: 6.5-8.5; oxygen concentration: > 80% of air saturation value; photoperiod: 12 h daylight / 12 h darkness
- Type and amount of food: fish were fed with live food of brine shrimp (O.S.I®)
- Feeding frequency during acclimation: fish were fed twice a day. Fish were not fed for 24 h before weighing at the start of the test
- Health during acclimation (any mortality observed): no diseases were observed during the acclimating period, no mortality was present within 7 d before test beginning
- Other: test fish of similar size were chosen before the test and anaesthesised with 80 mg/L MS-222. Fish were blotted dry and weighted. Then tehy were put into test water for 30 min for recovery.

Test type:
Water media type:
Limit test:
Total exposure duration:
28 d
165 mg CaCO3/L (treatment), 160 mg CaCO3/L (control)
Test temperature:
22.3 °C - 24.2 °C
6.94 to 7.80
Dissolved oxygen:
Oxygen cocentration: 70.6 % - 94.8 % of air saturation
Nominal and measured concentrations:
- control, 100 mg/L (nominal)
- both < LOD (89.6 ng/mL) (measured)
Details on test conditions:
- Test vessel: 18 L flass tanks
- Filled volume: 15 L
- Aeration: yes, gentle aeration
- Renewal rate of test solution (frequency/flow rate): twice per week
- No. of organisms per vessel: 20
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.1 g fish/L at the beginning of the test, 0.18 g fish/L at the end of the test

- Source/preparation of dilution water: tap water dechlorinated by activated carbon and sterilized by UV lamp was used and then aerated for 24 h.
- Total organic carbon: 746.91 µg/L
- Particulate matter: 3 mg/L
- Intervals of water quality measurement: hardness: once during the test; pH and dissolved oxygen: at the beginning, before and after each renewal and at the end of the test; temperature: daily

- Adjustment of pH: no data
- Photoperiod: 12 h light - 12 h dark
- Light intensity: no data

EFFECT PARAMETERS MEASURED : fish were inspected and recorded once a day for mortality and other effects. Dead fish were removed when observed and the feeding rate was adjusted

Reference substance (positive control):
Key result
28 d
Dose descriptor:
Effect conc.:
>= 100 mg/L
Nominal / measured:
Conc. based on:
other: test mat. (saturated solution)
Basis for effect:
Remarks on result:
other: Measured concentrations of the test substance were below the limit of detection (89.6 ng/mL)
Details on results:
- Mortality in the controls: none
- Other: the measured concentrations of the test substance were all below the limit of detection (LOD2=89.6 ng/mL)

Reported statistics and error estimates:
The variances among the means of the specific growth rate in both groups was analyzed by one-way ANOVA and the mean of treatment groups was compared with the control group using Homoscedastic t test. The data treatment was carried out with software TOXCALC (v 5.0.26).

The mean weight of fish after 28 d was: 0.1350 g in the control and 0.1294 g in the treatment. The specific growth rate of treatment group was not significantly different from that of the control (P > 0.05).

Both the EC50 value and the lowest observed effect concentration (LOEC) of growth rate for juvenile Gobiocypris rarus exposed to the test substance for 28 d was higher than the concentration of the satuated solution of the test substance.

Description of key information

NOEC (28 d) ≥ 100 mg/L, based on mortality ofGobiocypris rarus(OECD 215)

Key value for chemical safety assessment

Additional information

In the study by Jing (2014), the long-term toxicity of Reaction mass of 1,1'-(isopropylidene)bis[3,5-dibromo-4-(2,3-dibromo-2-methylpropoxy)benzene] and 1,3-dibromo-2-(2,3-dibromo-2-methylpropoxy)-5-{2-[3,5-dibromo-4-(2,3,3-tribromo-2-methylpropoxy)phenyl]propan-2-yl}benzene to fish was tested according to the OECD guideline 215 and GLP. Gobiocypris rarus was exposed 28 d in a semi-static setup to the test concentration of 100 mg/L (nominal). This concentration was chosen based on the results of an acute toxicity test on fish. As the test item is poorly soluble in water, the test item solution was stirred and filtered prior to test initiation, in order to prepare a saturated solution. The concentrations of the test substance during the test were monitored by UPLC. The measured concentrations of the test substance were all below the limit of detection (89.6 ng/mL). No mortalities or other effects were observed during the test and resulted in a NOEC (28 d) of≥100 mg/L (nominal).