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Long-term toxicity to fish

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Reference
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999-03-18 - 1999-04-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
equivalent or similar to
Guideline:
other: IBAMA - Portaria No. 84/96
Version / remarks:
A 168-hour chronic toxicity study of Ferbam to Zebrafish larvae {Brachydanio rerió) was conducted. Ten larvae fish with maximum of 24-hour old per test concentration plus dilution water and solvent controls were used in a nominal dosing regime of 5.6; 10; 32; 56; 100; 320; 560 and 1000 ug/L.
Deviations:
no
GLP compliance:
no
Analytical monitoring:
not specified
Details on sampling:
not specified
Vehicle:
yes
Details on test solutions:
Initial stock solution was prepared by dissolving 50 g of test material in 10 mL of dimethyl sulphoxide, then it was completed with reconstituted water to a total volume of 500 mL. To assist the solubilization five drops of Tween-80 and stirring for 10 minutes were used. All standard weights and dilutions are listed in the raw data.
Test organisms (species):
other: Brachydanio rerio
Details on test organisms:
Source of organism
The eggs of zebrafish were obtained at the Aquatic Biology Laboratory - BIOAGRI. The matrix fish to reproduction were obtained from reliable supplier (Peixe Vivo Aquicultura Ltda - Muriaé, MG), which has provided fish to BIOAGRI for six years.

Acclimation
These eggs were incubated in aquarium culture (40 L) under reconstituted natural water with adjusted pH to 7.0 ± 0.2, and hardness at 25 mg/L. It was maintained controlled environment conditions such as temperature to 28°C, and photoperiod 16 hour light and 8 hour darkness. Observations were made for at least 48 hours prior to testing during embryo incubation and hatching. One hour before the initiation of the test, larvae were been removed using a Pasteur pipette from the culture aquarium and placed into the chamber with dilution water.

Feeding
During the time of acclimation and exposure of test material the larvae fish were held without food.
Test type:
semi-static
Water media type:
other: reconstituted water composed of the following compounds in the amounts stated per liter of deionized water: 4.8 g NaHCO3, 1.5 g CaSO4, 6.1 g MgSO4 and 0.2 g KCl
Limit test:
no
Total exposure duration:
168 h
Post exposure observation period:
not specified
Hardness:
Start: 47.5 mg/L as CaCO3
48 hours: 51.0 mg/L as CaCO3
96 hours: 45.6 mg /L as CaCO3
144 hours: 43.35 mg/L as CaCO3
Test temperature:
Start: 22.3 °C
48 hours: 22.6 °C
96 hours: 22.9 °C
144 hours: 22.4 °C
pH:
Start: 7.60
48 hours: 7.54
96 hours: 7.58
144 hours: 7.54
Dissolved oxygen:
Start: 5.3 mg/L
48 hours: 5.0 mg/L
96 hours: 5.2 mg/L
144 hours: 5.0 mg/L
Salinity:
not specified
Conductivity:
Start: 155 ug/cm
48 hours: 162 ug/cm
96 hours: 163 ug/cm
144 hours: 147 ug/cm
Nominal and measured concentrations:
not specified
Details on test conditions:
Test water
The bioassay was conducted in glass chamber containing 1000 mL of reconstituted water composed of the following compounds in the amounts stated per liter of dionized water: 4.8 g NaHCO3, 1.5 g CaSO4, 6.1 g MgSO4 and 0.2 g KCl.

Test chamber
It was used 1.5 L glass chamber containing 1000 mL of diluted stock solution.

Test Procedure
An initial range finding test was carried out using ten per concentration level. The concentrations: 1; 10; 100 and 1000 ug/L were used to find the toxic range. In this trial, it was used a static system (without renewed test solution) for 168 hours of incubation with photoperiod of 16 hours of light
Once the range found, the nominal concentrations 5.6; 10; 32; 56; 100; 320; 560 and 1000 ug/L were selected to conducted a definitive test. The final test was then carried out as mentioned above, but using a semi-static system with renewed of test-solution each 48 hours and twenty larvae fish per concentration. Details of the iest solution and control solution preparations can be found on Raw data.
Control and solvent control in the same quantity used in the highest concentration, both were conducted as mentioned before.
The organisms were placed in each glass chamber according to arrangement (5+5).
Observations for mortality and sublethal/behavioral responses were made every 24 hours during the 168 hour test period. Dead individuals were removed at each observation period.

Reference test
The reference test using potassium dichromate was conducted in order to verify the sensibility of test-organism. It was carried out as mentioned above, in the final test, but using the following concentrations: 32; 56; 100; 120 and 160mg/L.
The LC50 values and 95% confidence limits were determined using the Trimmed Spearman-Karber Method.

Glassware preparation
All glassware prior the test, were washed with commercial detergent solution. After rinsed with tap water, they were washed with commercial acetone, sulphocromic solution, and then nitric acid 5% (v/v) to remove any residue. They were washed again in tap water followed by deionized water.

Statistical analysis method
Based on the data obtained from the definitive test, the LC50 and 95% confidence limits were determined using the Trimmed Spearman- Karber Method (HAMILTON et al.,1978) and Fishers Exact Test (TOXSTAT 3.3) was used to detect differences between the control and the surviving animals in each concentration tested.
Analysis of variance were used for significant test substance effects. The statistical tests employed and the results of these tests were reported in raw data. The criterion selected for MATC computation is the one which exhibits an effect ( a statistically significant difference between treatment and control groups: P< 0.05) to calculate the NOEC and LOEC values.
Reference substance (positive control):
yes
Key result
Duration:
168 h
Dose descriptor:
NOEC
Effect conc.:
100 µg/L
Nominal / measured:
not specified
Conc. based on:
other: tested concentrations
Basis for effect:
mortality
Key result
Duration:
168 h
Dose descriptor:
LC50
Effect conc.:
291.36 µg/L
Nominal / measured:
not specified
Conc. based on:
other: tested concentrations
Basis for effect:
mortality
Key result
Duration:
168 h
Dose descriptor:
LOEC
Effect conc.:
320 µg/L
Nominal / measured:
not specified
Conc. based on:
other: tested concentrations
Basis for effect:
mortality
Key result
Duration:
168 h
Dose descriptor:
other: MATC
Effect conc.:
178.88 µg/L
Nominal / measured:
not specified
Conc. based on:
other: tested concentrations
Basis for effect:
mortality
Details on results:
The highest level that had no significant lethal effect on the larvae (NOEC) was 100 ug/L. The lowest concentration that showed significant lethal effect (LOEC) was 320 ug/L in 168 hours of exposure. The MATC value was 178.88 ug/L
It was observed lethargic effect of Ferbam for the test organism to concentrations: 32; 56; 100; 320; 560 and 1000 ug /L.
Results with reference substance (positive control):
see 'Any other information on results incl. tables'

Table: Chronic toxicity of the test substance and a reference compound K2Cr2O7 to Zebrafish larvae (Brachydanio rerio)

Test substance LC50 in micrograms/Liter (ppb)
Period of incubation
24 48 72 96 120 144 168
Ferbam - - 999.99 613.04 461.08 45.47 371.82
K2Cr2O7* - - - 127.01 114.16 80.75 77.01
*ppm
Validity criteria fulfilled:
yes
Conclusions:
A 168-hour LC50 was calculated to be 291.36 ug/L. The highest level that had no significant lethal effect on the larvae (NOEC) was 100 ug/L. The lowest concentration that showed significant lethal effect (LOEC) was 320 ug/L in 168 hours of exposure. The MATC value was 178.88 ug/L
Executive summary:

A 168-hour chronic toxicity study of Ferbam to Zebrafish larvae {Brachydanio rerió) was conducted. Ten larvae fish with maximum of 24-hour old per test concentration plus dilution water and solvent controls were used in a nominal dosing regime of 5.6; 10; 32; 56; 100; 320; 560 and 1000 ug/L.

From the data collected during this study, a 168-hour LC50 with 95% confidence limits was calculated to be 291.36 (236.20 - 359.42 ug/L) based on the tested concentrations. For mortality the highest concentration that showed no significant effect (NOEC) was 100 ug/L and the lowest concentration that showed significant effect (LOEC) was 320 ug/L. The maximum acceptable toxicant concentration (MATC) value was 178.88 ug/L. Sublethal/behavioral response like as lethargic was noted among the larvae fish to concentrations 32; 56; 100; 320; 560 and 1000 ug/L. As a quality check the larvae sensibility tested with a reference compound potassium dichromate, which 168-hour LC50 value was 77.01 mg/L (70.92 - 83.62) mg/L.

Description of key information

Test procedure in accordance with generally accepted scientific standards and described in sufficient detail

no GLP

LC50: 291.36 µg/L

NOEC: 100 µg/L

LOEC: 320 µg/L

MATC: 178.88 µg/L

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater fish:
100 µg/L

Additional information

A 168-hour chronic toxicity study of Ferbam to Zebrafish larvae (Brachydanio rerio) was conducted. Ten larvae fish with maximum of 24-hour old per test concentration plus dilution water and solvent controls were used in a nominal dosing regime of 5.6; 10; 32; 56; 100; 320; 560 and 1000 µg/L.

From the data collected during this study, a 168-hour LC50 with 95% confidence limits was calculated to be 291.36 (236.20 - 359.42) µg/L based on the tested concentrations. For mortality the highest concentration that showed no significant effect (NOEC) was 100 ug/L and the lowest concentration that showed significant effect (LOEC) was 320 µg/L. The maximum acceptable toxicant concentration (MATC) value was 178.88 µg/L. Sublethal/behavioral response like as lethargic was noted among the larvae fish to concentrations 32; 56; 100; 320; 560 and 1000 µg/L. As a quality check the larvae sensibility tested with a reference compound potassium dichromate, which 168-hour LC50 value was 77.01 (70.92 - 83.62) mg/L.