Registration Dossier

Ecotoxicological information

Long-term toxicity to fish

Currently viewing:

Administrative data

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002

Materials and methods

Test material

Reference
Name:
Unnamed

Sampling and analysis

Details on sampling:
- Sampling method: From all vessels 2 samples were taken by the technical staff of the chemical laboratory in gastight, closed HPLC vials. The samples were analysed by HPLC directly after sampling.
- Sampling frequency: Up to day 11, daily samples were taken to monitor the test concentrations during the adjustment period of the final flow rates. From day 14 to day 35, the systems were sampled two times a week.

Test organisms

Details on test organisms:
TEST ORGANISM
- Common name: zebrafish
- Source: laboratory breed. Origin of the strain of zebrafish: West Aquarium GmbH, PB 146, 37431 Bad Lauterberg, Germany. Fertilised eggs for the test were obtained from individuals which were reared in the laboratory of the Fraunhofer-Institute, Schmallenberg, Germany.


METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish: approx. 80 fish per vessel. Holding temperature was 26 °C ± 1 °C. Light/dark cycle was 12 h/12 h. Flow through rate was adjusted to reach a 2-fold exchange of water per day. Fish were fed daily ad libitum with TetraMinR Hauptfutter (Tetra Werke, Melle, Germany) and brine shrimp nauplii (Artemia salina).
- Method of collection of fertilised eggs: Eggs were collected with a spawning-tray (made of glass) which was placed at the bottom of the holding vessels. The tray was covered with a lattice (stainless steel) to prevent the adults from predating on the eggs, and artificial plant substrate to stimulate spawning into the tray. Mating of the fish was induced, when lighting was switched on (one fluorescent lamp per vessel, light intensity approximately 1000 lux, measured 5 cm above the water surface in the middle of the test vessel).
- Subsequent handling of eggs: The collected eggs were transferred from the spawning-tray onto a sieve, rinsed with clean water in order to remove faeces and food residuals, and then put into glass dishes. Fertilised eggs (microscopic determination of early blastula stage) were then pipetted (widened and de-burred pipette tip) into the test solutions. Time from spawning until transferring into the test solutions did not exceed two hours.


POST-HATCH FEEDING
- Start date: from day 6
- Type/source of feed: from day 6 on with breeding food (Tetra, AZ 000). From day 9 on, brine shrimp nauplii (Artemia salina). From day 16 on ground TetraMin flake food was added to the daily food.
- Amount given: ad libitum
- Frequency of feeding:daily

Study design

Total exposure duration:
35 d

Test conditions

Test temperature:
25.7 (±0.7) °C
pH:
6.5 - 8.2 (depending on test concentrations)
Nominal and measured concentrations:
Test concentrations (nominal): 9.38, 18.8, 37.5, 75.0,  and 150 mg/L
Details on test conditions:
TEST SYSTEM
- Emybro cups (if used, type/material, size, fill volume): Fertilised eggs were kept in plastic cages with nylon nets forming the bottom of the cages.
- Test vessel:
- Type (delete if not applicable): closed by plexiglass covers
- Material, size, headspace, fill volume: full glass aquaria of 30 x 22 x 24 cm (l x w x h) with a total volume of 15.8 litres and approx. 10.0 litres of test solution
- Aeration: no
- Type of flow-through: Dilution water was pumped by membrane pumps for each vessel. Adequate amounts of stock solution were added via a 12 channel peristaltic pump. The test substance solution was introduced beneath the water surface.
- Renewal rate of test solution: 12 volumes per day
- No. of fertilized eggs/embryos per vessel: 100 fertilised eggs
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: purified drinking water according to OECD-guideline The purification included filtration with activated charcoal, passage through a lime-stone column and aeration. To avoid copper contamination, plastic water pipes are used for the testing facilities.
- Intervals of water quality measurement: pH-value, oxygen concentration, and temperature were measured directly before adding the fish and afterwards daily up to day 11 and in the following period twice a week until study termination. Minimum and maximum values of the water temperature were measured continuously and recorded together with the other measurements.


OTHER TEST CONDITIONS
- Photoperiod: light/dark cycle of 12 h/12 h


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Endpoints: mortality/survival rate, hatching rate, length and weight. After 6, 14, 21, and 35 days larvae/juvenile fish were photographed (digital camera: Olympus C 1400 L) and the survival rates (each date) as well as the lengths (at day 35, only) of the animals were determined using digital image processing (UTHSCSA ImageTool Version 2.0, Alpha 2; University of Texas Health Science Center at San Antonio, 1997).

Results and discussion

Effect concentrationsopen allclose all
Duration:
35 d
Effect conc.:
9.4 mg/L
Duration:
35 d
Effect conc.:
18.8 mg/L
Duration:
35 d
Effect conc.:
33.7 mg/L
Details on results:
Methyl methacrylate affected survival at concentrations equal to or  higher than 18.8 mg/L mainly at the life stage before, during and  directly after hatch (within the first 6 days). At the same  concentrations, growth was reduced significantly. Thus, after 35 days  (study termination), the NOEC (based on nominal concentrations) was  observed at 9.4 mg/L, the LOEC at 18.8 mg/L, the LC50 (based on  time-weighted averages) was calculated with 33.7 mg/L (c.l. 30.7-37.1 mg/L), the LC10 with 16.9 mg/L.

Any other information on results incl. tables

Results of the analytical measurements of the test concnetration:

 

Nominal concetrations (mg/L)

 

9.4

18.8

37.5

75

150

Total mean

9.04

22.1

50.0

114

214

% of nominal

96

118

133

152

143

Hatching success and survival up to day 6, post hatch success and total success (%) up to day 6

 

Nominal concentration (mg/L)

success

Control

9.4

18.8

37.5

75

150

Hatch

95

94

91

92

78

85

60

68

0

0

0

0

Post-hatch

86

83

88

83

90

79

37

37

 

 

 

 

Total

80

77

78

75

69

66

21

24

0

0

0

0

Weights and mean lengths at study termination

 

Nominal concentration (mg/L)

 

control

9.4

18.8

37.5

Group weights (g)

1.240

1.100

1.240

1.325

0.885

0.873

0.270

0.282

Weight per fish (mg)

15

14

16

17

13

13

12*

11*

Mean lenght (cm)

1.164

1.031

1.150

1.120

0.936*

0.993*

0.840*

0.883*

 significance p<0.05

Applicant's summary and conclusion

Conclusions:
In a valid guideline study, after 35 days, the NOEC (nominal concentrations) was  9.4 mg/L, the LOEC was 18.8 mg/L, the LC50 (based on time-weighted averages) was 33.7 mg/L  and the LC10 was 16.9 mg/L.