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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:
01/09/2000 - 25/09/2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD TG 210 (OPPTS 850.1400) in compliance with GLP, without deviations that influence the quality of the results.
Qualifier:
according to
Guideline:
EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: nominal: 0.02, 0.05, 0.2, 0.4 and 0.8 mg/l
- Sampling method: During the prolonged toxicity study, the following sampling regime was applied for confirmation of test concentrations based on measurement of radioactivity.

Number Semi-static period: Singular samples from each solution when freshly prepared and from each vessel when samples were taken from the spent solutions.
Flow-through period: from each vessel, i.e. two samples per concentration.
Frequency Semi-static period: on days 0, 1, 3, 4, 5, 6, 7, 8
Flow-through period: on days 9, 14, 16, 21, 28
Volume 2 ml

During the prolonged toxicity study, the following sampling regime was applied for confirmation of the stability of the test substance in the test solutions based on Thin Layer Chromatography (TLC).

Frequency On days 0, 8, 14, 21 and 28 samples were taken from each vessel, i.e. two samples per concentration. Per concentration the samples taken from the replicates were pooled before extraction.
Volume ca. 300 to 500 ml.
TLC conditions Stationary phase: Si-60 (20x20, Merck); mobile phase: acetone and n-hexane (95%) at a ratio of 70% and 30% (vtv), respctively. Two-dimensional TLC scanner: Electronic Autoradiography Instant Imager (Packard Instrument Company)
Samples actually analysed Day 0: target 0.8 mgn, Day 8: target 0.4 and 0.8 mg/I, Day 14: target 0.2, 0.4 and 0.8 mg/I, Day 21: target 0.2
and 0.4 mg/l.

- Sample storage conditions before analysis: samples for determining concentrations were measured on the day of sampling. Samples for determining test substance stability were stored in a deep-freeze until analysis.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The standard test procedures required generation of test solutions, which should contain completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that disturb the test system should be prevented (e.g. film of the test substance on the water surface).

The water solubility of OTNE is 2.68 mg/I. The density is 0.963 g/ml. Initially stock solutions were prepared at total OTNE concentrations of 0.4,1.0, 4.0, 8.0 and 16 mg/ml in acetone according to the protocol. However, this was amended in the first week of the test, when the stocks were diluted with acetone to concentrations, which were a factor of two lower. A volume of 0.10 ml of these adjusted stocks was added or dosed per liter test medium providing the required test concentrations. Each concentration consisted of a mixture of radio labelled OTNE-14C and unlabelled OTNE. The stock solutions were prepared daily. Until day 4 the test solutions were daily prepared by dilution of the stocks in test medium to renew the test solutions during the pre-hatching exposure in the petri-dishes. From day 4 to day 8 the solutions in the flow through vessels were with OTNE-14C (Flow-Through) daily spiked with freshly prepared solutions as the flow-through system failed to maintain the concentrations at an acceptable level.
- Controls: yes, blanks and vehicle controls
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 100 ppm, (100 ul/l)
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish
- Strain: No data
- Source: Ornamental fish hatchery Atlante, Hellevoetsluis, The Netherlands


METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs): No data, male/female ratio 1:1
- Method of collection of fertilised eggs: Collected from the bottom of the spawning tank which was covered with coconut hair which enables collection of the fertilised eggs without being eaten by the parent fish.
- Subsequent handling of eggs: No data


POST-HATCH FEEDING
- Start date: day 3
- Type/source of feed: Brachious until day 14, Artemia starting from day 11
- Amount given: Brachionus starting with 10 per larva, up to 200 per larva; Artemia starting with 15 per larva, up to 40 per larva
- Frequency of feeding: Brachionus starting with once a day, finally thrice a day; Artemia twice or thrice a day
Test type:
other: semi-static until day 4 after all eggs had hatched, then flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
30 d
Hardness:
250 mg/l CaCO3
Test temperature:
23.2 - 25.3 degrees Celsius
pH:
7.4 - 8.1
Dissolved oxygen:
6.0 - 8.8 mg/l
Nominal and measured concentrations:
Nominal: 0.02, 0.05, 0.2, 0.4 and 0.8 mg/l
Measured (day 0): 0.017, 0.038, 0.153, 0.311 and 0.536 mg/l
Measured (day 1 spent): 0.007, 0.047, 0.169, 0.242 and 0.659 mg/l
Measured (day 14 flow): 0.013, 0.032, 0.145, 0.236 and 0.174 mg/l
Measured (day 28 flow): 0.014, 0.035, 0.145 and 0.258 mg/l
For other concentrations see attached background material (data and results)
Details on test conditions:
TEST SYSTEM
- Emybro cups (if used, type/material, size, fill volume): sterile petri-dishes with a diameter of 10 cm until all larvae had hatched.Thereafter, the larvae were exposed in a flow-through system
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: stainless steel, 2 litres
- Aeration: No data
- Renewal rate of test solution (frequency/flow rate): 305 ml/h
- No. of fertilized eggs/embryos per vessel: 30
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 4


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: freshly prepared ultrapure water containing:
CaCl2.2H2O 293.8 mg/l
MgSO4.7H2O 123.3 mg/l
NaHCO3 64.8 mg/l
KCl 5.8 mg/l
- Total organic carbon: < 2 ppm
- Culture medium different from test medium: No
- Intervals of water quality measurement: three times a week


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16/8 light/dark
- Light intensity: 600-700 lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- stage of embryonic development (daily)
- hatching and survival (Daily from nominal day 3, numbers of hatched larvae and dead embryos, larvae and juvenile fish were recorded. Dead embryos, larvae and juvenile fish were removed directly after recording)
- criteria for death (Eggs: particularly in the early stages, a marked loss of translucency and change in coloration, caused by coagulation and/or precipitating of protein, characterised by a white opaque appearance; Embryos: Absence of body movement and /or absence of heartbeat; Larvae and juvenile fish: immobility and/or absence of respiratory movement and/or absence of heart-beat and/or white opaque coloration of the central nervous system and/or lack of reaction to mechanical stimulus)
- abnormal appearance (Daily, during the test, if present, abnormalities were recorded, e.g. hyperventilating, uncoordinated swimming, atypical quiescence and typical behaviour)
- body weigth and length (At the end of the test, all surviving fish were individually weighed and measured)



VEHICLE CONTROL PERFORMED: yes
Reference substance (positive control):
no
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
0.54 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: radioactive compound
Basis for effect:
mortality
Remarks:
egg survival
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
0.54 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: radioactive compound
Basis for effect:
time to hatch
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
0.3 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: radioactive compound
Basis for effect:
mortality
Remarks:
post hatch survival
Key result
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
0.16 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: radioactive compound
Basis for effect:
length
Remarks:
and weight
Duration:
30 d
Dose descriptor:
LOEC
Effect conc.:
0.29 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: radioactive compound
Basis for effect:
length
Remarks:
and weight
Duration:
30 d
Dose descriptor:
LC50
Effect conc.:
> 0.3 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: radioactive compound
Basis for effect:
length
Remarks:
and weight
Details on results:
- Mortality/survival at embryo, larval, juvenile, and adult stages: egg-survival (0-24h): 83-100%, larvae and embryo survival (0-14d): 83-97%; larvae survival (15-30d): 0-97% (no survival in highest concentration)
- Days to hatch or time to release of young: 43.6 - 79.6 hours in blanks.
- Observations on body length and weight of young and/or exposed parents at one or more time periods: after 30 days of exposure, OTNE has a significant effect on body weight/length of the hatched larvae (0.29 mg/l, measured).
- Other biological observations: OTNE did not induce any significant, visible effects on the development of zebra fish embryos at or below 0.544 mg/I (meas.)
- Effect concentrations exceeding solubility of substance in test medium: No
- Incidents in the course of the test which might have influenced the results: None
Reported statistics and error estimates:
DATA HANDLING

Determination of L(E)C50 values and respective parameters: If relevant, the LC50 was determined using the maximum likelihood estimation method with the probits of the percentages of dead fish as function of the logarithms of the corresponding concentrations (SAS v. 6.12) and the EC50 for sub-lethal effects by regression analysis.

Statistical anal is:
For each concentration the relevant data were tested for normality and for homogeneity of variance. Furthermore, these data were statistically tested using an ANOVA test followed by a mean comparison test (e.g. Tukey HSD test, Dunnett's t-test or Bonferroni's t-Test) or a rank test (e.g. Steel's Many-One Rank Test or Wilcoxon Rank Sum Test). When possible, the NOEC and LOEC for survival, hatching time and growth were determined on basis of these statistics. In addition the MATC was calculated as the geometric mean of the overall NOEC and LOEC.
Validity criteria fulfilled:
yes
Remarks:
Oxygen concentration >60% during test, water temperature within range criteria with some incidental deviations, test substance concentration varied more than 20% with no significant effect on TWA, survival for embryo's and post-hatch above 60 and 70% resp
Conclusions:
The lowest 30d-NOEC of OTNE towards the early life stages of Danio rerio is the NOEC for body weight and length at 0.16 mg/l. The 30d-LOEC and 30d-LC50 for body weight and length are 0.29 and >0.30 mg/l respectively.
Executive summary:

The toxicity of OTNE towards the early life stages of Danio rerio was investigated according to international guidelines (OPPTS 85.1400 / OECD Guideline 210) under GLP. Fish were exposed to 14C-labeled OTNE in a flow-through test (semi-static until day 4 after all eggs had hatched) at nominal concentrations of 0.02, 0.05, 0.2, 0.4 and 0.8 mg/l. Validity criteria for the test were met. The 30d-NOECs based on mean measured concentrations for egg survival, hatching time, post hatch survival and body weight and length were found to be 0.54, 0.54, 0.30 and 0.16 mg/l respectively. The 30d-LOEC and 30d-LC50 for body weight and length were found to be 0.29 and >0.30 mg/l respectively (also based on mean measured concentrations).

Description of key information

 The toxicity of OTNE towards the early life stages of Danio rerio was investigated according to international guidelines (OPPTS 85.1400 / OECD Guideline 210) under GLP. Fish were exposed to 14C-labeled OTNE in a flow-through test (semi-static until day 4 after all eggs had hatched) at nominal concentrations of 0.02, 0.05, 0.2, 0.4 and 0.8 mg/l. Validity criteria for the test were met. The 30d-NOECs based on mean measured concentrations for egg survival, hatching time, post hatch survival and body weight and length were found to be 0.54, 0.54, 0.30 and 0.16 mg/l respectively. The 30d-LOEC and 30d-LC50 for body weight and length were found to be 0.29 and >0.30 mg/l respectively (also based on mean measured concentrations). 

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater fish:
0.16 mg/L

Additional information