Registration Dossier

Ecotoxicological information

Long-term toxicity to fish

Currently viewing:

Administrative data

Endpoint:
fish early-life stage toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
02 Oct. - 13 Nov. 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD draft), limitation: poor stability of TS, estimation of most likely effect concentrations

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:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Version / remarks:
OECD Draft Guideline Fish, Early Life Stage Toxicity Test (1988)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): phenanthrene
- Molecular formula (if other than submission substance): C14H10
- Molecular weight (if other than submission substance): 178
- Substance type: organic
- Physical state: solid
- Source: Aldrich Chemie
- Analytical purity: >98 %
- Typical concentration in wash oil: 1.5 - 2.5 % (R4CC, GC analysis)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES (data from NL 2008: Coal-Tar Pitch, Annex-XV dossier, and WHO 1998: EHC 202, if not otherwise indicated)
- Vapour pressure: 1.6 - 2.6*10^-2 Pa (at 25 °C)
- Water solubility: in freshwater, approx. 970 - 1290 µg/L (20 - 25 °C)
- Henry's law constant: 3.7 Pa*m3/mol (at 25 °C)
- log Pow = 4.6

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/NL
- 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
- Type/source of feed: Immediately after hatching: 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).
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
- No. of vessels per concentration (replicates): 4
(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 80 fertilized eggs per test concentration,
thus to assure early exposure in the young blastula stage.)
- 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
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
ca. 11 µg/L
Nominal / measured:
estimated
Conc. based on:
dissolved
Remarks:
test material
Basis for effect:
larval development
Remarks on result:
other: calculated from the nominal concentration by dividing by 3
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
ca. 19 µg/L
Nominal / measured:
estimated
Conc. based on:
dissolved
Remarks:
test material
Basis for effect:
larval development
Remarks on result:
other: calculated from the nominal concentration by dividing by 3
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
ca. 150 µg/L
Nominal / measured:
estimated
Conc. based on:
dissolved
Remarks:
test material
Basis for effect:
mortality
Remarks:
of neonate fish
Remarks on result:
other: calculated from a mean nominal LC10 of 450 µg/L by dividing by 3
Duration:
28 d
Dose descriptor:
LC10
Effect conc.:
> 320 - < 560 µg/L
Nominal / measured:
nominal
Conc. based on:
dissolved
Remarks:
test material
Basis for effect:
mortality
Remarks:
of neonate fish
Remarks on result:
other: considered as NOEC
Duration:
28 d
Dose descriptor:
LC50
Effect conc.:
> 560 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Remarks:
test material
Basis for effect:
mortality
Remarks:
of neonate fish
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
32 µg/L
Nominal / measured:
nominal
Conc. based on:
dissolved
Remarks:
test material
Basis for effect:
larval development
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
56 µg/L
Nominal / measured:
nominal
Conc. based on:
dissolved
Remarks:
test material
Basis for effect:
larval development
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 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