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Ecotoxicological information

Long-term toxicity to aquatic invertebrates

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Administrative data

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 April 2016 to 15 June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000.
Qualifier:
according to
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Version / remarks:
Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000
Principles of method if other than guideline:
The test was performed on a water accomodated fraction
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Name in the study report: CaDNNSA
Description: Light brown powder with dark brown lumps
Batch:983-104
Purity: UVCB (treated as 100% pure)

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 0.46, 1.0, 2.2, 4.6 and 10 mg/L
- Sampling times: on day 0, 6, 9, 10 and 14 from fresh solutions; on day 2, 8, 10 and 16 from old solutions
- Sample storage conditions before analysis: stored in freezer (≤ -15°C). Storage stability of samples under these conditions was demonstrated in project 498817.
- On the day of analysis, the test samples were defrosted at room temperature. The samples were diluted in a 1:1 (v:v) ratio with acetonitrile and analysed. Calibration solutions were injected in duplicate. Test samples and QC samples were analysed by single injection.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:Preparation of test solutions started with individual loading rates ranging from 0.46 to 100 mg/L applying 2-day period of magnetic stirring. The obtained mixtures containing undissolved material were filtered through a 0.45 µm membrane filter (RC55, Whatman). All tested solutions were clear and colourless.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Daphnia magna (Crustacea, Cladocera) (Straus, 1820)
- Source: in-house culture
- Age of parental stock: at least third generation (2nd to 5th brood of parental daphnids 2-4 weeks of age)
- Feeding during test: algae suspension + marinure (ratio 5:1)
- Amount: 0.3 mg C/daphnia/day (adjusted in the weekend)
- Frequency: daily (and once per weekend)

mortality <20%, no presence of males, no ephippia or discoloured animals and there was no delay in the production of the first brood.

Study design

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

Test conditions

Hardness:
196-232 as mg CaCO3/L
Test temperature:
19-21°C
pH:
fresh solutions: 7.7-8.1
old solutions: 7.8-9.2
Dissolved oxygen:
fresh solutions: 8.3-9.4 mg/L
old solutions: 7.6-12 mg/L
Nominal and measured concentrations:
Nominal: 0, 0.46, 1.0, 2.2, 4.6 and 10 mg/L
Measured (average): NA, 0.0052, 0.17, 0.14, 0.20 and 0.28 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: all glass 60 mL
- Type: closed
- Aeration: no
- Renewal rate of test solution: every 48 hours (except on day 8 and 9 every 24 hours)
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 20

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M7, as prescribed by Dr. Elendt-Schneider
:Composition of medium M7 (TOC 0.051mg/L):
Adjusted ISO medium: the following chemicals (analytical grade) are dissolved in tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):
Macro salts: CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L
Medium M7: trace elements, macro nutrients and vitamins are added to freshly prepared ISO medium to reach the following concentrations:
Trace elements: B 0.125 mg/L
Fe 0.05 mg/L
Mn 0.025 mg/L
Li, Rb and Sr 0.0125 mg/L
Mo 0.0063 mg/L
Br 0.0025 mg/L
Cu 0.0016 mg/L
Zn 0.0063 mg/L
Co and I 0.0025 mg/L
Se 0.0010 mg/L
V 0.0003 mg/L
Na2EDTA.2H2O 2.5 mg/L
Macro nutrients:
Na2SiO3.9H2O 10.0 mg/L
NaNO3 0.27 mg/L
KH2PO4 0.14 mg/L
K2HPO4 0.18 mg/L
Vitamins: Thiamine 75.0 µg/L
B12 1.0 µg/L
Biotin 0.75 µg/L
The hardness: 180 mg/L expressed as CaCO3 and the pH: 7.7 ± 0.3.


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h daily
- Light intensity: at the start: 693-751 lux ; the end: 609-682 lux

EFFECT PARAMETERS MEASURED :
Parental
Number of living, immobile and dead parental daphnids; e very workday and upon renewal on non-workdays.
Presence of eggs in the brood pouch: every workday and upon renewal on non-workdays.
Body length: at the end of the test.
Offspring
Appearance first brood: Wwen observed.
Newborn daphnids (number and condition): every workday and upon renewal on non-workdays (young were removed).
Presence of unhatched eggs: when observed.
Incidence of immobility:when observed.
Test medium
Temperature, oxygen and pH: at the start of the test and just before and after each renewal in one of the vessels of each test group with surviving daphnids.
Hardness: weekly in fresh and old media from the control and the highest test concentration
Light: at the start and the end of the test


TEST CONCENTRATIONS
- Range finding study: yes 10 days at 0,1.0, 10 and 100 mg/L (renewal at day 1, 3 and 6); performed with WAF concentrations over 24 and 48 h 85-98% of initial, over 72 hours 60-71% of initial --> attributed to adsorption to algae
- Results used to determine the conditions for the definitive study: mortality of all parental daphnids at 100 mg/L; reduced reproduction at 10 mg/L
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
4.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
2.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
10 < mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: no dose response
Details on results:
- Observations on body length: significantly decreased at 4.6 and 10 mg/L
- Other biological observations: discoloration at 4.6 and 10 mg/L
- Parental mortality (immobility): 5, 10, 10, 30, 0 and 40% at 0, 0.46, 1.0, 2.2, 4.6 and 10 mg/L (in absence of a dose response no statistical significant effect could be assessed)
- Effect on reproduction:
mean cummulative number of juveniles per introduced parent: 166.1, 165.6, 170.7, 132.7, 155.2 and 113.5 at 0, 0.46, 1.0, 2.2, 4.6 and 10 mg/L
mean cummulative number of juveniles per surviving parent: 169.4, 182.0, 181.7, 157.1, 155.2 and 141.7 at 0, 0.46, 1.0, 2.2, 4.6 and 10 mg/L
- Day of first brood: no treatment related effects
Reported statistics and error estimates:
very extensive statistical evaluation of the results was performed leading to the following:
Effect parameters
Parameter Loading rate (mg/L)
NOEL for reproduction 4.6*
EL10 for reproduction 2.7 (0.46-16)* ; 5.2 (3.3-8.2)#
EL50 for reproduction >10
NOEL for mortality 10
NOEL for growth 2.2
() – 95% confidence intervals, * - based on introduced daphnids, # - based on surviving parents**- based on absence of dose-response

Applicant's summary and conclusion

Executive summary:

In a semi-static daphnia reproduction study (OECD 211) daphnids were exposed during 21 days at loading rates of 0, 0.46, 1.0, 2.2, 4.6 and 10 mg/L (WAF). Mean measured concentrations were NA, 0.0052, 0.17, 0.14, 0.20 and 0.28 mg/L. Effects on growth were reported at a loading rate of 2.2 mg/L. The NOEL for reproduction and mortality were 4.6 and 10 mg/L(expressed as loading rate).