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Ecotoxicological information

Long-term toxicity to fish

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Administrative data

Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 Oct. - 13 Nov. 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
guideline was a draft OECD test guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Version / remarks:
OECD Draft Guideline Fish, Early-life Stage Toxicity Test (1988)
Principles of method if other than guideline:
The study was performed similar to the OECD Draft TG 210 (Fish, Early-life Stage Toxicity Test). As limitation for the test, the poor stability/persistence of the test material within the renewal periods was noted. As consequence, the most likely effect concentrations were estimated based on variable measured concentrations by application of a correction factor.
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Phenanthrene
EC Number:
201-581-5
EC Name:
Phenanthrene
Cas Number:
85-01-8
Molecular formula:
C14H10
IUPAC Name:
phenanthrene
Test material form:
solid
Details on test material:
- Substance type: organic
- for additional information see study record
Specific details on test material used for the study:
- Name of test material (as cited in study report): phenanthrene
- Source of test material: Aldrich Chemie
- Analytical purity: > 98 %

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Sampling prior to start, on days 6, 13, 20 (first test) and 7, 14, and 21 (second test) in test solution just after preparation and just before replacement (see analytical results in Report, 3.6, Tab. 3, p. 15/16).

Test solutions

Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Aqueous stock solution with about 1 mg phenanthrene/L (saturated)
- Eluate: yes; test solutions were prepared by the elution column technique (results see Report 3.1, p. 11): Chromosorb was used as adsorbens, coated with an acetonic PHEN solution (about 1 % in acetone). Dilution water was purged through the steel column packed with the coated carrier material. Saturated solutions were diluted with dilution water for testing.
- Differential loading: no, prepared by sequential dilution from the stock
- Controls: dilution water

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: zebra fish
- Source: commercial hatchery M.B. Ruijsbrook B.V., Maassluis, The Netherlands
- Acclimatisation: ≥ 2 wks, 24 °C
- Housing: females individual, separated from males, 7 h light, 17 h dark

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs): 1 egg-carrying female placed into a basin with three males previously added (temp. 26 °C)
- Subsequent handling of eggs: deposition in the test vessels, microscopic observation of development

POST-HATCH FEEDING
- Start date: immediately after hatching
- Type/source of feed: from day 1 to 8 rotifers; from day 9 enriched with Artemia nauplii (enriched with Selco rotifiers)
- Frequency of feeding: daily

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
28 d

Test conditions

Hardness:
207 - 217 mg/L (as CaCO3)
Test temperature:
22.8 - 24.9 °C
pH:
7.2 - 8.2
Dissolved oxygen:
>= 5 mg/L, in isolated cases 3.5 - 5.0 mg/L
Nominal and measured concentrations:
nominal: 18, 32, 56, 100, 180, and 320 µg/L (1st test) / 320 and 560 µg/L (2nd test)
Average analytical values (18, 56, 180 µg/L not measured) were 72 - 113 % of nominal before renewal of the test medium, but were only 17 % to just above detection limit in the test medium to be replaced (high loss of TS). A general correction factor of 1/3 (nominal to analytical values) is derived from the availabe measured data.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 1L glass beaker with 800 mL of test solution
- Aeration: slight aeration
- Renewal rate of test solution (frequency/flow rate): at the first 3 days, the control and test solutions were replaced daily. During the remaining test duration, all solutions were renewed 3x/wk (Monday, Wednesday and Friday)
- No. of fertilized eggs/embryos per vessel: 20; at the start of the test, ca. 60 potentially fertilized eggs were placed in each test compartment (total ~240) and, after first exposure of 24 h, were reduced to about 20 fertilized eggs per vessel (total of 80 per test concentration), thus to assure early exposure in the young blastula stage.
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Groundwater supplemented with minerals
- Total organic carbon: 1.5 - 1.7 mg C/L
- Ca/Mg ratio: approx. 2:1

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light / 8 h dark (yellow light)
- Light intensity: no data

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
Hatching success, mortality, and malformations of eggs and larvae

VEHICLE CONTROL PERFORMED: yes
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
ca. 11 µg/L
Nominal / measured:
estimated
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
larval development
Remarks on result:
other: calculated from the nominal concentration by dividing by 3 (general correction factor to account for measured concentrations in the renewal solutions at the start and at the end of the renewal period)
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
32 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
larval development
Key result
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
ca. 19 µg/L
Nominal / measured:
estimated
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
larval development
Remarks on result:
other: calculated from the nominal concentration by dividing by 3 (general correction factor to account for measured concentrations in the renewal solutions at the start and at the end of the renewal period)
Key result
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
56 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
larval development
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
ca. 150 µg/L
Nominal / measured:
estimated
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mortality
Remarks:
neonate fish
Remarks on result:
other: calculated from a mean nominal LC10 of 450 µg/L by dividing by 3 (general correction factor to account for measured concentrations in the renewal solutions at the start and at the end of the renewal period)
Details on results:
The LC10-value can be regarded as the NOEC for mortality of larvae, and the estimate would be in this case be 450 µg/L (between 320 and 560 µg/L). The nominal values are lowered by a factor of 3 to approximate the effective concentrations.
Reported statistics and error estimates:
Mortality: binominal test at 95- and 98-% significance level
Growth: two-tailed Dunnett-test with 95- and 99-% significance level in both cases as compared to controls

Any other information on results incl. tables

Summary of the results on hatching and mortality of eggs, and mortality and growth of larvae of Brachydanio rerio exposed to several test concentrations of phenanthrene

Nominal conc. of phenanthrene (µg/L)

% of eggs hatched

% mortality after 28 d (eggs and larvae)

Length (cm) (larvae)

Wet weight (mg) (larvae)

0

100

1.3

1.17 ± 0.08

12.9 ± 2.21

18

100

2.5

1.16 ± 0.07

12.5 ± 2.13

32

98

1.2

1.15 ± 0.09

12.3 ± 2.53

56

100

0

1.141)± 0.08

12.4 ± 2.15

100

100

1.3

1.122) ± 0.08

11.72) ± 2.19

180

99

3.7

1.122) ± 0.08

11.52) ± 1.69

320

100

3.8

1.042) ± 0.07

9.712) ± 1.86

Second test

0

99

0

1.29 ± 0.08

14.8 ± 1.88

320

97

6.3

1.002) ± 0.12

7.332) ± 2.15

560

94

15

0.882) ± 0.11

4.402)± 1.61

1)Significantly less than control (two tailed Dunett-test, p=0.05)

2)Significantly less than control (two tailed Dunett-test, p=0.01)

There is a dose-related, statistically significant decrease in the length and weight of the fish, although the differences to the control are not large in absolute numbers. In the second test, the growth reduction in fish exposed to 320 µg/L was somewhat more pronounced than in the first test.

Applicant's summary and conclusion

Validity criteria fulfilled:
not specified
Conclusions:
In a study similar to OECD TG 210 (Fish, Early-life Stage Toxicity Test) (draft TG from 1988), a NOEC of 0.011 mg/L was determined for the development of newly hatched larvae of Danio rerio (previous name: Brachydanio rerio). The estimated NOEC for mortality of neonate fish was ca. 0.150 mg/L (highest concentration tested, mean value of two test series).