Registration Dossier

Ecotoxicological information

Endocrine disrupter testing in aquatic vertebrates – in vivo

Administrative data

Endpoint:
fish: other
Remarks:
fish sexual development
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Oct 16, 2018 – Dec 18, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD TG 234 (Fish Sexual Development Test)
Version / remarks:
28.07.2011
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
impurity
Type:
Constituent
Test material form:
solid: granular

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
At test start, after 7 days and after 14 days, samples were taken from all test vessels in order to confirm correct dosing. During the remaining course of the study, samples were taken on a weekly basis, alternating between the vessels that were served by the same dosage pump. At test end, samples of all vessels were analysed.

Test solutions

Vehicle:
no

Test organisms

Aquatic vertebrate type:
fish
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish
- Strain:
- Source: Test facility bred (origin: West Aquarium GmbH, 37431 Bad Lauterberg, Germany)

- Life stage:
- Age at study initiation (mean and range, SD): fertilized eggs
- Length at study initiation (length definition, mean, range and SD):
- Weight at study initiation (mean and range, SD):
- Method of breeding:
- Pre-exposure reproductive information

ACCLIMATION
- Acclimation period:
- Acclimation conditions (same as test or not):
- Type and amount of food during acclimation:
- Feeding frequency during acclimation:
- Health during acclimation (any mortality observed):

QUARANTINE (wild caught)
- Duration: n/a
- Health/mortality: n/a

FEEDING DURING TEST
- Food type: TetraMin® Hauptfutter (Tetra Werke, Melle, Germany) and brine shrimp nauplii (Artemia salina)
- Amount: ad libitum
- Frequency: daily

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs):
- Method of collection of fertilised eggs: glass spawning-tray covered with a stainless steel lattice
- Subsequent handling of eggs: The collected eggs were transferred from the spawning-tray into a sieve, rinsed with clean water in order to remove any debris and then put into glass dishes. Fertilized eggs (microscopic determination of > four cell stage, i.e. early blastula stage) were transferred by means of a widened and de-burred pipette tip into the test chambers. Time from spawning until transferring into the test solutions was kept as short as possible and no later than 12 h after fertilization.
- Subsequent handling of juvenile fish:

POST-HATCH FEEDING
From day 7 on (dpf = days post fertilization), larvae were fed once daily with ground breeding food (TetraMin Baby, Tetra Werke, Melle, Germany). From day 14 (dpf) on, brine shrimp nauplii (Artemia salina) were added once daily. From the same date on, ground TetraMin flakes were added once daily to the fish feed.

Study design

Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
63 d

Test conditions

Hardness:
1.2-1.4 mmol/L
Test temperature:
27.0 °C ± 2.0 °C
pH:
6.26-7.24
Dissolved oxygen:
87-117 %
Conductivity:
295 – 323 µS/cm
Nominal and measured concentrations:
nominal: 0.10 0.32, 1.00, 3.20, 10.0 mg/L (main vessels); 1.00, 10.0 mg/L (add. vessels)
measured: 0.104, 0.331, 1.07, 3.34, 11.0 mg/L (main vessels); 1.11, 10.9 mg/L (add. vessels)
Details on test conditions:
TEST SYSTEM
- Test vessel: aquaria
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass, 28 L total volume, 25 L test solution
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter): membrane pump
- Renewal rate of test solution (frequency/flow rate): 10 volumes per vessel and day
- No. of organisms per vessel: 30 eggs at test start
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Vehicle control performed: no
- No. of vessels per vehicle control (replicates): n/a
- Biomass loading rate:
- Stocking density:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: purified drinking water (filtration with activated charcoal and aeration)
- Total organic carbon: 0.3985 – 0.6134 mg/L (DOC)
- Particulate matter: n/a
- Metals (cadmium, chrome, copper, iron, manganese, nickel, lead and zinc): < 15 µg/L (sum parameter)
- Pesticides: n/a
- Chlorine: 0.02 mg/L
- Alkalinity: 1.8 – 2.2 mmol/L
- Ca/mg ratio: 1:2
- Culture medium different from test medium: no
- Intervals of water quality measurement: monthly

OTHER TEST CONDITIONS
- Adjustment of pH: yes, (37 % HCl) via dosage system
- Photoperiod: 12/12 light/dark
- Light intensity: approx. 1000 lumen

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
3 dpf: Time to hatch, Hatching success
21 dpf: post-hatch survival
35 dpf: post-hatch survival, length
63 dpf: survival, length and weight, sex ratio, gonad histopathology, VTG content

TEST CONCENTRATIONS
- Spacing factor for test concentrations:√10
- Justification for using less concentrations than requested by guideline:

RANGE FINDING STUDY (test was performed following the basic requirements of OECD guideline 210)
- Test concentrations: 0.1, 1.0, 10 mg/L
- Results used to determine the conditions for the definitive study: No effects on survival were observed.
Reference substance (positive control):
no

Results and discussion

Effect concentrations
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
1.07 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: post-hatch survival
Remarks on result:
other: preliminary result
Results with reference substance (positive control):
n/a

Applicant's summary and conclusion

Validity criteria fulfilled:
yes