Registration Dossier

Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
uvcb substance
Specific details on test material used for the study:
uvcb substance

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: nominal loading rate: 0, 1.98 - 2.96 - 4.45 - 6.67 - 10.0 mg/L
- Sampling method:
All loading rates and the control were analyzed three times during the test (once within a period of 7 days) in the fresh media at the start of an exposure-renewal interval (0 hours, on test days 0, 7 and 14) as well as in the old media at the end of an exposure-renewal interval (24 hours, on test days 1, 8, 15).
Three aliquots were taken as samples after preparation of the WAFs. Two replicates of them were stabilized immediately by addition of acetonitrile containing 0.2% formic acid and stored at ambient conditions until analysis. The third replicate was prepared without addition of the solvent and analyzed.

- Sample storage conditions before analysis: at ambient conditions until analysis

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: five water accommodated fractions (WAFs) were prepared with nominal loading rates of the test item of: 1.98 - 2.96 - 4.45 - 6.67 - 10.0 mg/L (separation factor: 1.5). For each loading rate, an appropriate amount of the test item was pipetted onto the surface of an appropriate amount of the dilution water. For this purpose, the density was taken into account. A slow stirring procedure was applied for 72 ± 2 hour at room temperature. After a separation phase of 30 minutes at room temperature, the aqueous phase or WAF was removed by siphoning (from the approximate bottom of the glass flask).The resulting WAFs were used for testing.
- Controls: 10 replicates of dilution water without test item (negative control).
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The WAFs were checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). The Tyndall effect was negative in all WAFs and the control throughout the exposure period.
- Other relevant information: A coating phase (saturation of the test container) was carried out. The test containers were pre-treated with the appropriate test solution for at least 12 hours under test conditions. Before the start of the exposure, the test containers were emptied and refilled with freshly prepared test solution.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Strain/clone: Daphnia magna STRAUS
- Age at study initiation (mean and range, SD): Less than 24 hours old daphnids from a healthy stock. To achieve this, juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within this period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.
- Method of breeding: In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20  2°C, in an incubator, 16 hours illumination; light intensity of max. 1500 lx., Elendt M4, according to OECD 211
- Source: obtained from continuous laboratory cultures. The daphnids of the laboratory culture at the test facility originated from clone 5, Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), Berlin, Germany
- Feeding during test : Daily feeding with unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, max. 0.2 mg C / daphnia and day

ACCLIMATION
- Acclimation period: Acclimatization was not necessary, because the composition of the dilution water is equivalent to the culture medium.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS:
Feeding of the culture stocks: The daphnids are fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus. The algae are cultured at the test facility.

Study design

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

Test conditions

Hardness:
mg/L CaCO3
Control: fresh medium: 255-277, old medium: 274-291
Highest loading rate (WAF 10 mg/L): fresh medium: 254-282, old medium: 258-274
Test temperature:
°C
Control: fresh medium: 19.5-21.6, old medium: 20.1-21.4
Highest loading rate (WAF 10 mg/L): fresh medium: 19.9-21.5, old medium:20.2-21.1
pH:
Control: fresh medium: 7.40-7.67, old medium: 7.14-7.46
Highest loading rate (WAF 10 mg/L): fresh medium: 7.30-7.62, old medium: 7.13-7.40
Dissolved oxygen:
mg/L
Control: fresh medium: 7.72-8.29, old medium: 3.68-8.54
Highest loading rate (WAF 10 mg/L): fresh medium: 8.12-8.22, old medium: 5.84-8.82
Nominal and measured concentrations:
nominal loading rate: 0, 1.98 - 2.96 - 4.45 - 6.67 - 10.0 mg/L (separation factor: 1.5)
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beakers (5 (ID) x 8 (H) cm), 100 mL capacity, loosely covered with watch glasses.
- Fill volume: 50mL
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 10 daphnids and 10 replicates were used for all WAFs and the control. 1 daphnid was held individually per replicate.
- No. of vessels per concentration (replicates): 10/test concentration
- No. of vessels per control (replicates): 10/control

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4, according to OECD 211, pH 8.2
- Culture medium different from test medium: no
- Intervals of water quality measurement:
pH-values, dissolved oxygen concentration, total hardness and water temperature were measured once within 7 days, in the fresh media at the start of an exposure-renewal interval (0 hours) as well as in the old media at the end of an exposure-renewal interval (24 hours), in one replicate of the control and the highest loading rate of the test item with a living parent animal. The water quality parameters in the fresh media were measured in an additional replicate without daphnids of the highest loading rate of the test item and the control. At the end of the exposure-renewal interval, the water quality parameters of the old media were measured in a test vessel of the highest loading rate of the test item and the control, which contains daphnids and food algae. The incubator temperature (measurement in air with a thermo-hygrograph) was recorded throughout the period of the test.
Acid capacity and acidity of the dilution water are determined at least quarterly and were reported.

OTHER TEST CONDITIONS
- Photoperiod: 16/8 hours light/dark
- Light intensity: Max. 1500 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Mortality of parental Daphnids: The number of parental daphnids, which were immobilised or dying during the test and the dates of death were observed and recorded once a day at least at the same time as the number of offspring was counted. Dead specimens were removed as soon as they were detected. Accidental mortality did not occur.
- Offspring: Trials were checked daily for first appearance of juveniles. The number of neonates (alive and dead progeny) was counted and the dates of release were recorded once a day from the first day of production of neonates until the end of the exposure. The neonates (alive and dead) were removed after counting and before addition of algae to prevent them from consuming food intended for the adults. The number of aborted eggs or dead offspring and the dates of observation were recorded. At the end of the test (21 days), the total number of living offspring was assessed.
- Additional observations /Condition of parental daphnids: Abnormalities (e.g. swimming behaviour, number of males and winter eggs) were observed and recorded on each day of observation.
- Total body length / Mean dry weight of parental daphnids: At the end of the test, the total length excluding the anal spine of each survived parental daphnid and the mean dry weight of the survived parental daphnids of all WAFs and the control were determined.
- Additional endpoints: The time to first brood and the number and size of first brood per animal were reported, but not used for endpoint calculations.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.5
- Range finding study: 12 days, 5 Daphnia (1 per replicate)
- Test concentrations range finding test: A control and three WAFs with nominal loading rates of the test item of 0.100, 1.00 and 10.0 mg/L
- Results used to determine the conditions for the definitive study: After 12 days no adult mortality was observed in the control and all nominal loading rates but at highest test concentration aborted eggs juveniles were observed.

STATISTICAL EVALUATION:
Significant differences of reproduction were determined in comparison to the control using statistical standard procedures as normality test (Shapiro-Wilk’s test), variance homogeneity test (Levene’s test), Step-down Jonckheere-Terpstra test procedure for reproduction. The Fisher’s exact binomial test with Bonferroni correction was used for adult mortality.
Prior to running the multiple test , a normality test and a variance homogeneity test were performed. P values of the normality and the variance homogeneity test were 0.01. The alpha-value (acceptable probability of incorrectly concluding that there is a difference) was alpha = 0.01 for the normality test and the variance homogeneity test.
For the effects on reproduction, the juveniles per introduced parent at the start of the test excluding accidental and inadvertent mortality (reproductive output) were counted. Since no accidental or inadvertent mortality was observed, no parental daphnids were excluded from the evaluation of the reproductive output.
The coefficients of variation around the mean number of living offspring produced per introduced parent and per survived parent in the control and the test groups were evaluated.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
21 d
Dose descriptor:
EL10
Effect conc.:
4.12 mg/L
Nominal / measured:
nominal
Conc. based on:
other: nominal loading rate (WAF)
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
other: nominal loading rate
Basis for effect:
reproduction
Remarks on result:
other: empirically derived
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
4.45 mg/L
Nominal / measured:
nominal
Conc. based on:
other: nominal loading rate (WAF)
Basis for effect:
reproduction
Details on results:
The average number of living juveniles at the end of the test after 21 days per introduced parental female daphnid was 96.9 in the control group.
Accidental and inadvertent mortality as defined in the OECD test guideline were not observed.
Cochran-Armitage trend test revealed a significant increasing trend (p ≤ 0.05) in adult mortality, the evaluation was done based on the cumulative offspring per introduced (female) parent.
Thus, according to OECD 211 it is required to report the effect values for the cumulative offspring per introduced parent provided these values are lower than in the cumulative offspring per survivor.
Reproduction:
A statistically significant reduction of the reproductive output in comparison to the reproductive output in the control was determined at the nominal loading rates of 6.67 and 10.0 mg/L. Step-down Jonckheere-Terpstra test procedure (α = 0.05) was used for statistical analysis of the reproductive output per introduced parent animals. Therefore the LOELR was determined to be 6.67 mg/L and the NOELR 4.45 mg/L for the cumulative offspring per introduced parent.
The determination of the ELR10-value and its 95% confidence limits was carried out by point estimates from the 3 parametric normal CDF.
The ELR50 could not be calculated by the software, since no reduction of the reproductive output greater than 50% occurred. The ELR50-value was empirically derived from the observed data for the reproductive output per introduced parent.
The derivation of NOELR/LOELR and ELR10-values is valid based on the above-mentioned statistical procedures. However, the reproduction rate of all parent Daphnia which died was comparable to the reproduction rate of surviving parental Daphnia. In addition, reproduction rates of treated Daphnia were also similar to the control group’s reproduction rate. Therefore, it can be assumed that a major reason for lower overall reproduction is the mortality of the parent Daphnia.
Mortality:
The test item induced adult mortality of 50% after 21 days in the nominal loading rate 10.0 mg/L and 10% in the loading rates 2.96 – 4 45 – 6 67 mg/L. However, OECD guideline 211 concedes a mortality rate of control Daphnia up to 20%; NOELR and LOELR were empirically derived.
NOELR adult mortality: 6.67 mg/L nominal loading rate (WAF)
LOELR adult mortality: 10 mg/L nominal loading rate (WAF)
First Appearance of living Juveniles
The first appearance of living juveniles was observed between days 7 and 9 at the introduced parental daphnids of the control and the nominal loading rates of 1.98 to 10.0 mg/L of the test item.
Stillborn Juveniles and Aborted Eggs
Aborted eggs were observed in the loading rates of 1.98 to 10.0 mg/L during the exposure period of 21 days. In the loading rates of 4.45 to 10.0 mg/L stillborn juveniles were observed.
- Growth (Total Body Length and Mean Dry Weight) of the Survived Parental Daphnids
At the end of the test Tthe mean values of the body length (excluding the anal spine) of the survived parental female daphnids in the tested loading rates of 1.98 to 10.0 mg/L were in the range of 4.0 to 4.3 mm per daphnid and 4.2 mm per female daphnid in the control group.
On day 21 Tthe mean dry weight of the survived parental daphnids was in the range of 0.78 to 0.90 mg per daphnid in the WAF loading rates of 1.98 to 10.0 mg/L and 0.71 mg per daphnid in the control
- Presence of Males
No males were observed in the control or in any of the test groups during the test.
- Occurrence of Ephippia (Winter Eggs)
No ephippia were observed in the control or in the test groups during the test.
- Analytical results
LC-MS/MS Analysis
Only the monoalkylated isomers could be quantified. These measured concentrations were either extremely low (1.36 – 4.65 µg/L) or generally even below the limit of quantification (<1 μg/L). It was concluded that these results could not be used to base test concentration upon. As a consequence, results of the present study were based on the loading rates initially prepared.
All reasonable efforts were taken to produce a saturated solution of all soluble components of the test substance in test media. Since the test substance is a multicomponent mixture (UVCB), the test solution is considered a water accommodated fraction (WAF). The term “loading rate” is advocated to express exposure to a WAF and is considered analogous to the nominal concentration. According to OECD guidance document No. 23 (2019), for tests with chemicals that cannot be quantified by analytical methods at the concentrations causing effects, the effect concentration can be expressed based on the nominal concentrations or loading rate (for mixtures).

Results with reference substance (positive control):
immobility for the reference item CAS 7778-50-9 after 24h: EC50 2.00mg/L (most recent of monthly test)
0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000)

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
No parent animal in the control group died. Mortality of the parent animals in the control group was 0% (< 20%). The mean number of living offspring produced per survived parent animal control after 21d 96.9 (>60).