Registration Dossier
Registration Dossier
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EC number: 271-867-2 | CAS number: 68610-51-5
Long-term toxicity to aquatic invertebrates
Administrative data
Link to relevant study record(s)
- Endpoint:
- long-term toxicity to aquatic invertebrates
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 08 May 2017 to 29 May 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 211 (Daphnia magna Reproduction Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Specific details on test material used for the study:
- No further details specified in the study report.
- Analytical monitoring:
- yes
- Details on sampling:
- Samples were taken from three replicates at the applied test concentration level and one replicate from the control at the following time points:
1. week: on day 0 (fresh media, start of the experiment) and on day 1 (old media)
2. week: on day 9 (fresh media) and on day 10 (old media)
3. week: on day 20 (fresh media) and on day 21 (old media, end of the experiment)
Additional samples (3 x 10 mL) were taken also (then frozen and kept approximately at -20°C at the Test Facility) and retained as a back-up, if required for any confirmatory analyses (discarded after satisfactory results were obtained on the first set of). - Vehicle:
- no
- Details on test solutions:
- Because the test item is poorly soluble in water, a test solution was prepared using a saturated solution method (water accommodated fraction, WAF) according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23.
Saturated test item solution (100 mg/L nominal loading rate WAF) was prepared by dispersing/dissolving the amount of test item into the test medium (ISO Medium) two days before the start of the test. This solution was shaken for about 24 hours at approximately 30°C and then equilibrated for about 24 hours at approximately 20°C. The non-dissolved test material was removed by filtration through a fine (0.22 μm) filter to give appropriate 100 mg/L nominal loading rate WAF. The maximum amount of test material in the medium has been exposed applying this procedure. Since the test was performed under semi-static conditions, prior to the treatment, at each of the renewal periods, test item solution was prepared by the method described above.
Test solutions renewals were performed at 24-hour intervals during the experiment. - Test organisms (species):
- Daphnia magna
- Details on test organisms:
- Species and strain: Daphnia magna Straus
Source: István Szent University, 2100 Gödöllő, Páter Károly u. 1, Hungary
Stock culture: Cultures was used for the test do not contain ephippia.
Breeding Conditions: Daphnia magna are bred in Ecotoxicological Laboratory of CiToxLAB Hungary Ltd.
Justification of strain: The Daphnia magna is the standard species of reproduction test to be used according to the OECD guideline 211.
Number of animals: Ten animals were tested in every concentration and in the control as well during the main experiment.
Loading: Parent animals were maintained individually, one per test vessel, with ~80 mL of test solution in each vessel.
Age of animals: Less than 24 hours old at the beginning of the test and not first brood progeny.
Acclimatisation: To avoid the necessity of adaptation prior to the test the quality of water used was similar to the culture media.
Parent animals: The animals present at the start of the test and their reproductive output was the object of this study.
Offspring: Produced by the parental animals in the course of this test. The parental animals were 8-10 days old during the test when the first young Daphnids emerged from the brood pouch, after which a new batch appears every two to three days generally. These batches are called "broods" of the F1 (filial 1) generation.
Water: For culturing Daphnia, Elendt M4 media was used.
Food and Feeding
The Daphnia were fed daily with concentrated algal suspension of Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum). The supplied diet was based on the amount of organic carbon (C) provided to each parent animal. The constant ration level was 0.15 mg C/Daphnia/day during the test.
The cell number of algal-suspension fed was counted before the test. The total organic carbon content (TOC) of algal-suspension fed was based on the results of the last annual nomographical TOC measurement. - Test type:
- semi-static
- Water media type:
- freshwater
- Limit test:
- yes
- Total exposure duration:
- 21 d
- Post exposure observation period:
- No post exposure observation period specified in the study report.
- Hardness:
- Hardness was above 140 mg/L (as CaCO3).
- Test temperature:
- The temperature of the test media and in the climate chamber was within the range 18 - 22°C, and the deviation from the mean temperature value was less than ± 2°C.
- pH:
- The pH was within the range 6 - 9, it did not vary by more than 1.5 units during the study period.
- Dissolved oxygen:
- The dissolved oxygen concentration was above 3 mg/L at the beginning and during the test.
- Salinity:
- Not specified
- Conductivity:
- Not specified
- Nominal and measured concentrations:
- Because significant effect was not observed at the examined concentration level during the 10-days preliminary range-finding test, a limit test was carried out using 100 mg/L loading rate WAF and one control were investigated in the definitive test.
- Details on test conditions:
- PERFORMANCE OF THE TEST
This 21-days reproduction test was carried out in a semi-static renewal system, with a renewal frequency of 24 hours. The frequency of test solutions renewal was based on the stability of the test substance and performed preliminary test.
When the medium was renewed a second series of test vessels were prepared and the parent animals transferred to them by a pipette of suitable diameter. The volume of test medium transferred with the Daphnia was minimised.
Observations
Mortality of the parent animals was recorded daily during the test. The living offspring produced by each parent animal were removed and counted daily from the appearance of the first brood.
Measurement of the length of the parent animals (excluding the anal spine) was performed at the end of the test.
Test conditions
A light-dark cycle was necessary: 8 hours darkness and 16 hours light. The light intensity was within the range of 15-20 μE m-2s-1 and it was checked at the start of the test.
Temperature, oxygen concentration and pH values were measured at the start of the experiment, thereafter at each renewal period in fresh and old media, in the control and in the test item concentration in one replicate.
Hardness was determined in fresh and old media, in the control as well as in the test item concentration at each renewal period. The temperature of the test media and in the climate chamber was within the range 18 - 22°C, and the deviation from the mean temperature value was less than ± 2°C. The dissolved oxygen concentration was above 3 mg/L at the beginning and during the test. The pH was within the range 6 - 9, it did not vary by more than 1.5 units during the study period.
Test vessels were not aerated during the test.
Hardness was above 140 mg/L (as CaCO3). - Reference substance (positive control):
- no
- Key result
- Duration:
- 21 d
- Dose descriptor:
- NOEC
- Effect conc.:
- 100 other: mg/L nominal loading rate WAF
- Nominal / measured:
- nominal
- Conc. based on:
- other: saturated solution
- Basis for effect:
- reproduction
- Details on results:
- MORTALITY OF PARENTS
Only one dead animal was observed in the test item treated group of 100.0 mg/L nominal loading rate WAF during the whole experiment. Mortality was not observed in the control.
JUVENILE PRODUCTION
The mean number of live offspring per parent animal surviving in the control at the end of the test was 151.40 with a variation coefficient of 5.63 %.
Due to the observed mortality (only one animal) in the test item group (100.0 mg/L WAF), one replicate has not been taken into account for calculation of juvenile production, but has been taken for the results of appearance of the first brood.
The time to production of first brood was the same in the test item group of 100.0 mg/L WAF as in the control group during the test.
The reproduction of the surviving parent animals was not significantly different from the control group in the examined test item treated group of 100.0 mg/L loading rate WAF (2 Sample t-Test, α = 0.05).
Statistically significant differences was not observed between the reproductive output of the parent animals per day at the test item treated group and the control (2 Sample t-Test, α =0.05) group.
Aborted eggs, dead offspring, males or ephippia were not found during the study.
BODY LENGTHS OF PARENT ANIMALS
The body length of the surviving parent animals in the test item treated group was not significantly different from the control group (2 Sample t-Test, α =0.05). - Results with reference substance (positive control):
- None
- Reported statistics and error estimates:
- Statistically significant difference was not observed concerning the body length of the surviving parent animals either (2 Sample t-Test, α = 0.05).
- Validity criteria fulfilled:
- yes
- Conclusions:
- In the 21-days Reproduction test on Daphnia magna with LOWINOX® CPL the 21-day LC50-value of parents and the 21-day reproduction EC10, EC20 and EC50 value were determined to be higher than 100.0 mg /L nominal loading rate WAF (considered to be a saturated solution). The NOEC for reproductive effect was determined to be 100.0 mg/L nominal loading rate and the LOEC was determined to be as higher than this tested level.
- Executive summary:
The purpose of the study was to estimate the effect of the test item LOWINOX® CPL on the reproductive output of Daphnia magna determining the NOEC, LOEC, EC50, EC20 and EC10 for reproduction after 21 days of exposure.
Daphnia magna were exposed in a semi-static test to saturated aqueous test media containing the test item at 100.0 mg/L nominal loading rate WAF in a limit test for 21 days.
The test method of application and the test species Daphnia magna are recommended by the test guidelines.
The 21-days reproduction test was carried out in a semi-static renewal system, with a renewal frequency of 24 hours based on analytical method validation (Study Code: 16/451-316AN) (due to that test item is a UVCB substance with very poor solubility).
Concentration of the test item in the test solution was analysed by the validated method - when freshly prepared and at renewal - on one occasion during the first week then these determinations were repeated weekly intervals thereafter based on the performed analytical method validation. Fully defined Elendt M4 medium was used as dilution water for the test solution.
Test item concentration could not be detected and therefore measured concentration was below the Limit of Detection (LOD = 0.75 μg/mL) for all WAF samples (freshly prepared and at renewal) during the experiment. The biological results are based on the nominal loading rate WAF. The maximum amount of test material in the medium has been exposed applying this procedure.
Test item exposure and control group comprised 10 Daphnids. The test animals were maintained individually, one per test vessel, with ~80 mL of test solution in each vessel.
The time to the production of first brood and the numbers of broods per animal were recorded during the test. The measurement of the length of the surviving parent animals was performed at the end of the test.
The average cumulative number of young per parent animal in the control was 151.40 with a variation coefficient of 5.63 %.
Only one dead animal was observed in the test item treated group of 100.0 mg/L nominal loading rate WAF during the whole experiment. Mortality was not observed in the control group.
The reproduction of the surviving parent animals was not significantly different from the control group in the test item treated group of 100.0 mg/L nominal loading rate WAF (2 Sample t-Test, α = 0.05).
Statistically significant difference was not observed concerning the body length of the surviving parent animals either (2 Sample t-Test, α = 0.05).
The time to production of first brood was the same in the test item group of 100.0 mg/L WAF as in the control group during the test.
In this 21-days Reproduction test on Daphnia magna with LOWINOX® CPL the 21-day LC50-value of parents and the 21-day reproduction EC10, EC20 and EC50 values were determined to be higher than 100.0 mg /L nominal loading rate WAF (considered to be a saturated solution). The NOEC for reproductive effect was determined to be 100.0 mg/L nominal loading rate and the LOEC was determined to be as higher than this tested level.
- Endpoint:
- long-term toxicity to aquatic invertebrates
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 28 October 2014 to 01 April 2015
- Reliability:
- 3 (not reliable)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- Study performed in accordance with OECD test guidelines in compliance with GLP. Testing conducted in accordance with ECHA Decision number CCH-D-0000003873-68-05/F. There is some doubt as to the definitive EC10 and NOELR values derived from the study demonstrating effects (the 21-day Daphnia study), as limit values of <1 mg/l where obtained for the reproduction endpoints. Given the deficiencies in the analytical method it is not clear as to whether these results are accurate. It was scientifically not possible to derive a lower value than this, due to the UVCB nature and low water solubility of the substance. It is the intetion of the registrant to repeat this study.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 211 (Daphnia magna Reproduction Test)
- Deviations:
- yes
- Remarks:
- See Any other information for details
- GLP compliance:
- yes
- Specific details on test material used for the study:
- Details on properties of test surrogate or analogue material (migrated information):
None specified - Analytical monitoring:
- yes
- Details on sampling:
- Samples for analysis were taken in triplicate from the test concentration and singular samples were taken from the control. In addition, the filter used to prepare the WAF was kept for possible analysis of the residue.
Sampling:
Frequency: At the start and the end of two intervals of 48 hours (nominal days 0 and 2, 7 and 9) and at the start of one interval of 72 hours (nominal day 16). After this day no more samples were taken.
Volume: A sample volume of 20 ml was taken and spiked into a separation funnel containing 20 ml dichloromethane.
Storage: Not applicable, samples were analysed on the day of sampling.
At the end of the refreshment period, the replicates were pooled at each concentration before sampling. - Vehicle:
- no
- Details on test solutions:
- The batch of LOWINOX® CPL tested was a UVCB and its appearance was described as a white to cream coloured powder. The substance was not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test substance.
All test concentrations were prepared separately and started with loading rates of 1.0, 10 and 100 mg/l for the range-finding test and with a loading rate of 1.0 mg/l for the reproduction test. Two days of magnetic stirring at room temperature in the dark was applied to reach the maximum solubility of the test substance in the test medium. The resulting aqueous mixtures were filtered through a 0.45 µm membrane filter (Whatman) where after the clear and colourless Water Accommodated Fractions (WAFs) were used as the test concentrations. - Test organisms (species):
- Daphnia magna
- Details on test organisms:
- Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.
Source: In-house laboratory culture with a known history.
Reason for selection: This system has been selected as an internationally accepted invertebrate species.
Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
Characteristics: To initiate the test, young daphnids < 24 hours old were selected, from parental daphnids greater than two weeks old.
Breeding
Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing them individually in 50 ml M7-medium (reproduction test) or by placing about 250 of them into 5 litres of medium in an all-glass culture vessel (range-finding test).
Maximum age of the cultures: 4 weeks
Monitoring of the individual cultures: Three times a week the young are counted and the parental daphnids are transferred to new media.
Renewal of the bulk cultures: After 7 days of cultivation half of the medium twice a week.
Temperature of medium: 18-22 °C
Feeding: Daily, a suspension of fresh water algae
Validity of the cultures: Historical data on the reproductive capacity are based on the numbers of living young counted three times a week in the individual cultures and tested to meet the validity criteria for survival and reproduction.
Medium M7, as prescribed by Dr. Elendt-Schneider
(Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).
Composition of medium M7:
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. - Test type:
- semi-static
- Water media type:
- freshwater
- Limit test:
- no
- Total exposure duration:
- 21 d
- Post exposure observation period:
- No post exposure observation period specified in the study report.
- Hardness:
- Total hardness varied between 179 and 232 mg calcium carbonate per litre, and thus complied to the requirements as laid down in the protocol (>140 mg CaCO3 per liter).
- Test temperature:
- The temperatures in the test media varied between 19 and 21°C. The temperature continuously measured in a temperature control vessel varied between 20 and 22°C during the test, and complied with the requirements as laid down in the protocol (18-22°C, constant within 2°C).
- pH:
- The pH remained within the range of 7.8 to 8.5 throughout the test and thus was maintained within the limits prescribed by the protocol (6.0-9.0, constant within 1.5 units).
- Dissolved oxygen:
- The oxygen concentration in all test solutions remained within the range of 7.5 to 10 mg/l during the exposure period and thus complied to the requirements as laid down in the protocol (>3 mg/l).
- Salinity:
- Not specified
- Nominal and measured concentrations:
- Concentrations in the test samples measured until day 9 of exposure were below the concentration of the lowest calibration solution, i.e. below 0.5 µg/l, except in one of the triplicate samples taken from the freshly prepared solution on day 7. In this sample, the concentration was measured to be 2.5 µg/l. Because this concentration was only found in one of the triplicate samples, it was assumed to be introduced during sample pretreatment.
Filters used during preparation of the test solutions were also analyzed. This confirmed that LOWINOX® CPL had been used for preparation.
No more samples were taken and analyzed during the remainder of the test period based on the results of the samples taken until day 16 which, showed that the actual test concentration was either below or around the concentration of the lowest calibration solution. The effect parameters were therefore based on the loading rate rather than on the measured test concentration. - Details on test conditions:
- Range-finding test
A range-finding test was performed to provide information about the range of concentrations to be used in the reproduction test. The concentrations of LOWINOX® CPL tested were: WAFs prepared at loading rates of 1.0, 10 and 100 mg/l. A control group was also included. Each concentration consisted of two replicates containing a total of ten daphnids (five each). The total test period was seven days. Test conditions were held as similar as possible to those applied in the reproduction test including feeding. Test solutions were renewed on days 1, 3 and 6 during the test. Singular samples for possible analysis were taken at days 0 (fresh), 1 (fresh and old), 3 (fresh and old) and 6 (old). Samples were taken from solutions containing the daphnia and food (algae), but also from an extra vessel incubated under the same conditions, but without daphnia and algae.
Reproduction test
Test concentrations
LOWINOX® CPL: WAF prepared at a loading rate of 1.0 mg/l
Control Test medium without test substance or other additives
Test procedure and conditions
Test duration: 21 days
Test type: Semi-static
Frequency of renewal: Three times a week
Test vessels: Volume: 60 ml (6 x Ø 3.5 cm), all-glass covered with a Perspex plate.
Medium: M7
Experimental design: At the start of the experiment (nominal day 0) 20 neonate daphnids, less than one day old, per group were divided over twenty vessels each containing a minimum of 50 ml test medium.
Light: 16 h photoperiod daily; intensity at the start: 778-825 lux; intensity at the end: 843-863 lux
Feeding: Twice daily an amount of 0.25 ml of Chlorella pyrenoidosa suspension. On weekend days an amount of 0.50 ml was added in one single feed. This daily ration corresponded to 0.2 mg C/Daphnia/day, which is the recommended value for daily feeding per daphnid in the reproduction test according to the OECD Guideline 211. From day 14 onwards, the total daily amount was increased to 0.75 ml, based on expertise. This daily ration corresponded to 0.3 mg C/Daphnia/day.
Measurements and recordings
Parental daphnids
Condition: Every workday and upon renewal on non-workdays, the number of living, immobile and dead parental daphnids was recorded. Dead daphnids were removed when observed.
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: When observed.
Neonate daphnids: Every workday and upon renewal on non-workdays, the number of neonates was counted and the condition of the young recorded. Thereafter the 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.
Hardness: Once a week in fresh and old media from each test group
Light: At the start and the end of the test - Reference substance (positive control):
- not specified
- Duration:
- 21 d
- Dose descriptor:
- NOELR
- Effect conc.:
- < 1 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- other: WAF loading rate
- Basis for effect:
- reproduction
- Duration:
- 21 d
- Dose descriptor:
- EL10
- Effect conc.:
- < 1 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- other: WAF loading rate
- Basis for effect:
- reproduction
- Duration:
- 21 d
- Dose descriptor:
- EL50
- Effect conc.:
- > 1 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- other: WAF loading rate
- Basis for effect:
- reproduction
- Duration:
- 21 d
- Dose descriptor:
- NOELR
- Effect conc.:
- 1 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- other: WAF loading rate
- Basis for effect:
- mortality
- Duration:
- 21 d
- Dose descriptor:
- NOELR
- Effect conc.:
- 1 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- other: WAF loading rate
- Basis for effect:
- growth
- Details on results:
- Range-finding test
No mortality was observed at any of the test concentrations and the control. On the first day(s) of exposure some of the daphnids exposed to the 10 and 100 mg/l WAFs were observed to be floating at the surface.
The presence of eggs in the brood pouch was generally recorded for the first time on day 6 for the control and the test concentrations. After 7 days of exposure almost all daphnids in the control and the test concentrations carried eggs in their brood pouch. Therefore, no effects on survival or time of egg development was observed at any of the WAFs when compared to the control.
Test conditions during the range-finding test were maintained within the limits prescribed by the protocol.
Samples taken from the WAFs prepared at 10 and 100 mg/l were analysed but the analytical method used proved to be insufficient, i.e. the method was not sensitive enough to measure the actual test concentrations in the test medium. It was however obvious that the actual concentrations in the WAFs were low, i.e. <0.01 mg/l.
After the performance of the range-finding test, the analytical method needed to be re-developed in order to measure lower test concentrations. It was decided to perform the reproduction test with only one test concentration, i.e. a WAF prepared at a loading rate of 1.0 mg/l, since WAFs prepared at higher test concentrations would only introduce the risk of having undissolved test material in the test vessels. Preparation of WAFs below 1.0 mg/l is technically not feasible.
Reproduction test
Measured concentrations
Concentrations in the test samples measured until day 9 of exposure were below the concentration of the lowest calibration solution, i.e. below 0.5 µg/l, except in one of the triplicate samples taken from the freshly prepared solution on day 7. In this sample, the concentration was measured to be 2.5 µg/l. Because this concentration was only found in one of the triplicate samples, it was assumed to be introduced during sample pretreatment.
Filters used during preparation of the test solutions were also analyzed. This confirmed that LOWINOX® CPL had been used for preparation.
No more samples were taken and analyzed during the remainder of the test period based on the results of the samples taken until day 16 which, showed that the actual test concentration was either below or around the concentration of the lowest calibration solution. The effect parameters were therefore based on the loading rate rather than on the measured test concentration.
Condition of parental daphnids
No parental daphnids in the control group died during the test period. Hence, parental mortality did not exceed 20% in the controls. A total number of four daphnids, i.e. 20%, died in the WAF prepared at a loading rate of 1.0 mg/l. No significant mortality was observed in the WAF when compared to the control.
In the control, the presence of eggs in the brood pouch was recorded for the first time on day 5 and the first brood appeared on day 8. On average the first recording of young was similar to the control in the WAF prepared at a loading rate of 1.0 mg/l.
Reproduction
On average, 91 young were produced per surviving daphnid in the control. Statistical analysis showed that the reproduction of the daphnids was significantly reduced in the WAF prepared at a loading rate of 1.0 mg/l.
Body length
Statistically significant reduction of growth was found in the WAF prepared at a loading rate of 1.0 mg/l. However, the effect was only 3% and therefore not considered biologically significant.
Experimental conditions
The pH remained within the range of 7.8 to 8.5 throughout the test and thus was maintained within the limits prescribed by the protocol (6.0-9.0, constant within 1.5 units).
The oxygen concentration in all test solutions remained within the range of 7.5 to 10 mg/l during the exposure period and thus complied to the requirements as laid down in the protocol (>3 mg/l).
The temperatures in the test media varied between 19 and 21 °C. The temperature continuously measured in a temperature control vessel varied between 20 and 22 °C during the test, and complied with the requirements as laid down in the protocol (18-22 °C, constant within 2 °C).
Total hardness varied between 179 and 232 mg calcium carbonate per litre, and thus complied to the requirements as laid down in the protocol (>140 mg CaCO3 per liter).
The total dissolved organic carbon content of the M7 medium was 0.22 mg/l. This complied with the requirements as laid down in the guidelines (TOC <2 mg/l). - Results with reference substance (positive control):
- Reference substances not required for this study.
- Reported statistics and error estimates:
- The NOELR for parental mortality was determined using Fisher’s Exact Binominal Test with Bonferroni Correction.
The following statistical procedures were used to determine the NOELR for reproduction and growth:
Data distribution: Shapiro-Wilk´s Test
Homogeneity of variance: Levene´s Test (with Residuals)
Differences between treatments and the control: Welch-t test for Inhomogeneous Variances (for reproduction) and Student-t test for Homogeneous Variances (for growth).
All analyses were performed with ToxRat Professional 2.10.05 (ToxRat Solutions® GmbH, Germany). - Validity criteria fulfilled:
- yes
- Conclusions:
- Under the conditions of the present study LOWINOX® CPL statistically significantly reduced the number of offspring of Daphnia magna by 21% in a WAF prepared at a loading rate of 1.0 mg/l after 21 days of exposure. Hence, the NOELR lies <1.0 mg/l. There is some doubt as to the definitive EC10 and NOELR values derived from the study demonstrating effects (the 21-day Daphnia study), as limit values of <1 mg/l where obtained for the reproduction endpoints. Given the deficiencies in the analytical method it is not clear as to whether these results are accurate. It was scientifically not possible to derive a lower value than this, due to the UVCB nature and low water solubility of the substance. It is the intention of the registrant to repeat this study, as the effects noted are not as expected, and required further clarification. This test will be repeated to further clarify these results and a spontaneous update to the registration completed.
- Executive summary:
Testing conducted in accordance with ECHA Decision number CCH-D-0000003873-68-05/F
According to the integrated testing strategy, the Daphnia study was to be conducted first. If based on the results of the long-term Daphnia study and the application of a relevant assessment factor, no risks are observed (PEC/PNEC<1), no long-term fish testing may need to be conducted. However, if a risk is indicated, the long-term fish study needs to be conducted.
Daphnia magna, 21-day reproduction study with LOWINOX® CPL.
The study procedures described in this report were based on the OECD guidelines for Testing of Chemicals: Guideline No. 211, 2012. In addition, the procedures were designed to meet the test methods and validity criteria of the ISO International Standard 10706, 2000, the Commission Regulation (EC) No 440/2008 Part C.20, 2008 and the OECD guidance document number 23, 2000.
The batch of LOWINOX® CPL tested was a UVCB and its appearance was described as a white to cream coloured powder. The substance was not completely soluble in test medium at the loading rates initially prepared.
A reproduction test was performed based on the results of a preceding 7-day range-finding test. Preparation of the test concentration started with a loading rate of 1.0 mg/l. Two days of magnetic stirring at room temperature in the dark was applied to reach the maximum solubility of the test substance in the test medium. The resulting aqueous mixture was filtered through a 0.45 µm membrane filter where after the clear and colourless Water Accommodated Fraction (WAF) was used as the test concentration.
The reproduction test was performed in a semi-static system, included 20 vessels for the test concentration and 20 vessels for a control group. Each of the vessels contained one neonate (<24h old) Daphnia magna in 50 ml test medium. The test duration was 21 days and the test solutions were renewed three times a week. The daphnids were fed on a daily basis with a Chlorella pyrenoidosa suspension. Every workday the condition of the parental daphnids was recorded, during the reproduction phase the number of living offspring, immobile young and appearance of unhatched (aborted) eggs was recorded. At the end of the test the length of the surviving parental daphnids was measured.
During the test, samples for analytical confirmation of actual exposure concentrations were taken at the start and the end of two intervals of 48 hours and at the start of one interval of 72 hours. Concentrations in the test samples measured until day 9 of exposure were generally below the Limit of Quantification (LOQ of 1 µg/l). Filters used during preparation of the test solutions were also analyzed. This confirmed that LOWINOX® CPL had been used for preparation.
No more samples were taken and analyzed during the remainder of the test period based on the results of the samples taken until day 9 which, showed that the actual test concentration was either below or around the LOQ. The effect parameters were therefore based on the loading rate rather than on the measured test concentration.
The study met the acceptability criteria prescribed by the protocol and was considered valid.
LOWINOX® CPL statistically significantly reduced the number of offspring of Daphnia magna by 21% in a WAF prepared at a loading rate of 1.0 mg/l after 21 days of exposure. Hence, the No Observed Effect Loading Rate (NOELR) lies <1.0 mg/l.
Effect parameters obtained in this study are summarized in the table belowParameter
Loading rate1(mg/l)
NOELR for reproduction
<1.0
EL10 for reproduction
<1.0
EL50 for reproduction
>1.0
NOELR for mortality
1.0
NOELR for growth
1.0
1WAF prepared at the given loading rate
Under the conditions of the present study LOWINOX® CPL appeared to statistically significantly reduced the number of offspring of Daphnia magna by 21% in a WAF prepared at a loading rate of 1.0 mg/l after 21 days of exposure. Hence, the NOELR lies <1.0 mg/l. There is some doubt as to the definitive EC10 and NOELR values derived from the study demonstrating effects (the 21-day Daphnia study), as limit values of <1 mg/l where obtained for the reproduction endpoints. Given the deficiencies in the analytical method it is not clear as to whether these results are accurate. It was scientifically not possible to derive a lower value than this, due to the UVCB nature and low water solubility of the substance.
For the purposes of risk assessment and on the following grounds:
1) There are no effects at the limit of solubility in water in acute toxicity studies.
2) There are no effects in the chronic fish test at the limit of solubility in water.
3) Effects are limited to reproduction only in the chronic daphnia study.
The value is set to 1 mg/l for the NOELR.
It is the intention of the registrant to repeat this study, as the effects noted are not as expected, and required further clarification. This test will be repeated to further clarify these results and a spontaneous update to the registration completed.
Referenceopen allclose all
Results of the Preliminary Range-Finding Test
Nominal loading rate WAF [mg/L] |
Untreated control |
100 |
Number of treated / immobilised animals |
5 / 0 |
5 / 0 |
Number of living newborns [mean] |
13.4 |
12.2 |
Reduction (%) |
- |
9.0 |
Mortality of parent animals (Daphnia magna)
Parameter (Day 21) |
Results (mg/L loading rate WAF, nominal) |
LC10 |
>100.0 |
LC20 |
>100.0 |
LC50 |
>100.0 |
NOEC (Mortality) |
100.0 |
LOEC (Mortality) |
>100.0 |
Juvenile reproduction of parent animals (Daphnia magna) (number of living newborns during the test)
Parameter (Day 21) |
Results (mg/L loading rate WAF, nominal) |
Reproduction EC10 95% conf. limits |
>100.0 (could not be determined)* |
Reproduction EC20 95% conf. limits |
>100.0 (could not be determined)* |
Reproduction EC50 95% conf. limits |
>100.0 (could not be determined)* |
NOEC (Reproduction) LOEC (Reproduction) |
100.0 >100.0 |
*Remark: As a limit test was performed the EC50value (and EC20or EC10as well) was not calculated and determined from the raw data.
Summary of reproductive output of the parent animals
Test group |
Mean number of living offspring per surviving parent animal |
SD |
CV (%) |
Reduction (%) |
Significance |
Control 100.0 mg/L WAF (nominal) |
151.40
136.67 |
8.53
7.50 |
5.63
5.49 |
-
9.7 |
-
n.s. |
SD: standard deviation
CV (%): variation coefficient
n.s.: statistically not significantly different compared to the control (2 Sample t-Test, α=0.05)
Mean body length of the survival parent animals at the end of the test
Test group |
Mean body length of parents surviving at the end of the test (mm) |
SD |
CV (%) |
Reduction (%) |
Significance |
Control 100.0 mg/L WAF (nominal) |
4.34
4.10 |
0.20
0.19 |
4.50
4.72 |
-
5.5 |
-
n.s. |
SD: standard deviation
CV (%): variation coefficient
n.s.: statistically not significantly different compared to the control (2 Sample t-Test, α=0.05)
Water Temperature, pH, Dissolved Oxygen of the test solutions
Time (day) |
Media |
Time (day) |
|||||
Control |
100.0 mg/L WAF |
||||||
°C |
pH |
mg/L |
°C |
pH |
mg/L |
||
0 |
Old |
- |
- |
- |
- |
- |
- |
Fresh |
20.3 |
7.59 |
8.5 |
20.3 |
7.75 |
7.7 |
|
1 |
Old |
20.4 |
7.81 |
8.5 |
20.4 |
8.22 |
9.2 |
Fresh |
20.5 |
7.57 |
8.3 |
20.4 |
7.79 |
7.3 |
|
2 |
Old |
20.3 |
7.81 |
8.7 |
20.3 |
8.08 |
9.2 |
Fresh |
20.6 |
7.66 |
8.5 |
20.5 |
7.71 |
7.4 |
|
3 |
Old |
20.2 |
7.81 |
8.7 |
20.4 |
8.07 |
9.0 |
Fresh |
20.5 |
7.63 |
8.2 |
20.5 |
7.76 |
7.2 |
|
4 |
Old |
20.4 |
7.85 |
8.5 |
20.4 |
8.05 |
8.8 |
Fresh |
20.6 |
7.66 |
8.5 |
20.6 |
7.79 |
7.3 |
|
5 |
Old |
20.3 |
7.77 |
8.7 |
20.3 |
7.85 |
8.6 |
Fresh |
20.7 |
7.68 |
8.4 |
20.5 |
7.79 |
7.4 |
|
6 |
Old |
20.4 |
7.99 |
8.9 |
20.4 |
7.92 |
8.3 |
Fresh |
20.6 |
7.82 |
8.4 |
20.6 |
7.78 |
7.4 |
|
7 |
Old |
20.4 |
7.98 |
8.9 |
20.4 |
8.08 |
9.0 |
Fresh |
20.6 |
7.80 |
8.4 |
20.6 |
7.82 |
7.2 |
|
8 |
Old |
20.5 |
7.98 |
8.9 |
20.5 |
7.94 |
8.8 |
Fresh |
20.6 |
7.72 |
8.4 |
20.6 |
7.89 |
7.63 |
|
9 |
Old |
20.5 |
7.93 |
8.8 |
20.4 |
8.05 |
8.8 |
Fresh |
20.6 |
7.69 |
8.2 |
20.5 |
7.86 |
7.4 |
|
10 |
Old |
20.4 |
7.88 |
8.9 |
20.5 |
7.88 |
8.9 |
Fresh |
20.6 |
7.62 |
8.5 |
20.6 |
7.71 |
7.9 |
|
11 |
Old |
20.4 |
7.81 |
8.5 |
20.4 |
7.91 |
8.5 |
Fresh |
20.5 |
7.69 |
8.1 |
20.5 |
7.81 |
7.4 |
|
12 |
Old |
20.5 |
7.84 |
8.8 |
20.5 |
7.94 |
8.7 |
Fresh |
20.7 |
7.73 |
8.1 |
20.6 |
7.80 |
7.5 |
|
13 |
Old |
20.7 |
7.80 |
8.6 |
20.8 |
7.92 |
8.7 |
Fresh |
20.8 |
7.73 |
8.4 |
20.8 |
7.83 |
7.3 |
|
14 |
Old |
20.5 |
7.89 |
8.6 |
20.5 |
7.98 |
8.6 |
Fresh |
20.7 |
7.78 |
8.5 |
20.5 |
7.87 |
7.2 |
|
15 |
Old |
20.5 |
7.81 |
8.8 |
20.5 |
7.90 |
8.7 |
Fresh |
20.8 |
7.79 |
8.4 |
20.8 |
7.88 |
7.2 |
|
16 |
Old |
20.4 |
7.71 |
8.7 |
20.4 |
7.86 |
8.7 |
Fresh |
20.7 |
7.76 |
8.3 |
20.8 |
7.89 |
7.7 |
|
17 |
Old |
20.3 |
7.64 |
8.5 |
20.4 |
7.77 |
8.6 |
Fresh |
20.6 |
7.63 |
8.3 |
20.6 |
7.71 |
7.2 |
|
18 |
Old |
20.2 |
7.82 |
8.5 |
20.3 |
7.87 |
8.6 |
Fresh |
20.7 |
8.26 |
8.4 |
20.7 |
7.90 |
7.3 |
|
19 |
Old |
20.4 |
7.90 |
8.4 |
20.4 |
7.92 |
8.6 |
Fresh |
20.7 |
8.24 |
8.5 |
20.7 |
7.84 |
7.4 |
|
20 |
Old |
20.5 |
7.97 |
8.5 |
20.5 |
7.94 |
8.4 |
Fresh |
20.6 |
8.13 |
8.4 |
20.7 |
8.02 |
7.3 |
|
21 |
Old |
20.3 |
7.70 |
8.4 |
20.3 |
7.74 |
8.5 |
Fresh |
- |
- |
- |
- |
- |
- |
|
Remark:°C = Temperature values
pH = pH values
mg/L = Dissolved oxygen values
Temperature values (°C) in the climate chamber
Day |
Minimum |
Maximum |
0 |
20.0 |
20.6 |
1 2 3 4 5 6 7 |
20.0 20.1 20.1 20.3 20.1 20.3 20.2 |
20.7 20.8 20.7 20.8 20.8 20.9 20.9 |
8 9 10 11 12 13 14 |
20.2 20.1 20.2 20.1 20.1 20.0 20.1 |
20.8 20.7 20.9 20.8 20.9 20.6 20.8 |
15 16 17 18 19 20 21 |
19.9 20.1 19.9 19.8 20.2 20.2 20.2 |
20.6 20.8 20.8 20.8 20.8 20.8 20.8 |
0-21 |
19.8 |
20.9 |
Water hardness (mg/L as CaCO3) values of the test solutions
Week |
Time Day |
Media |
Concentration |
|
Control |
100.0 mg/L WAF |
|||
1 |
0 |
Fresh |
259 |
262 |
1 |
Old Fresh |
263 259 |
262 261 |
|
2 |
Old Fresh |
255 253 |
252 272 |
|
3 |
Old Fresh |
271 285 |
248 283 |
|
4 |
Old Fresh |
273 286 |
250 286 |
|
5 |
Old Fresh |
259 273 |
252 278 |
|
6 |
Old Fresh |
261 282 |
258 279 |
|
7 |
Old Fresh |
253 270 |
247 278 |
|
2 |
8 |
Old Fresh |
263 276 |
259 272 |
9 |
Old Fresh |
257 269 |
261 275 |
|
10 |
Old Fresh |
262 277 |
268 273 |
|
11 |
Old Fresh |
258 275 |
260 271 |
|
12 |
Old Fresh |
265 279 |
258 271 |
|
13 |
Old Fresh |
266 278 |
257 270 |
|
14 |
Old Fresh |
265 281 |
260 274 |
|
3 |
15 |
Old Fresh |
267 284 |
262 273 |
16 |
Old Fresh |
267 281 |
265 275 |
|
17 |
Old Fresh |
269 277 |
263 271 |
|
18 |
Old Fresh |
278 249 |
274 270 |
|
19 |
Old Fresh |
272 251 |
255 277 |
|
20 |
Old Fresh |
269 277 |
252 272 |
|
21 |
Old |
263 |
257 |
|
Min. |
Old Fresh |
247 249 |
||
Max. |
Old Fresh |
278 286 |
||
Mortality of the parents at Control
Day |
Replicate |
|||||||||
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
|
1 2 3 4 5 6 7 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
8 9 10 11 12 13 14 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
15 16 17 18 19 20 21 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
Total Mortality (%) |
0 0.0 |
|||||||||
CD |
- |
|||||||||
Remark:CD = Deviation for the mean of control (%)
Mortality of the parents at 100.0 mg/L nominal loading rate WAF
Day |
Replicate |
|||||||||
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
|
1 2 3 4 5 6 7 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
8 9 10 11 12 13 14 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
15 16 17 18 19 20 21 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 1 - - - - |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
0 0 0 0 0 0 0 |
Total Mortality (%) |
1 10.0 |
|||||||||
CD |
10.0 |
|||||||||
Remark:CD = Deviation for the mean of control (%)
The Number of Juveniles at Control
Day |
Replicate |
|||||||||
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
|
1 2 3 4 5 6 7 |
0 0 0 0 0 0 16 |
0 0 0 0 0 0 19 |
0 0 0 0 0 0 21 |
0 0 0 0 0 0 17 |
0 0 0 0 0 0 16 |
0 0 0 0 0 0 19 |
0 0 0 0 0 0 19 |
0 0 0 0 0 0 12 |
0 0 0 0 0 0 17 |
0 0 0 0 0 0 15 |
8 9 10 11 12 13 14 |
0 0 20 0 0 36 0 |
0 0 36 0 0 35 0 |
0 0 39 0 0 37 0 |
0 0 44 0 0 39 0 |
0 0 41 0 0 47 0 |
0 37 1 0 34 0 0 |
1 0 36 0 0 34 0 |
0 0 37 0 0 39 1 |
0 0 38 0 0 42 0 |
1 0 33 0 0 39 0 |
15 16 17 18 19 20 21 |
0 31 0 0 31 0 0 |
0 33 0 0 30 0 0 |
0 30 0 0 31 0 0 |
0 28 0 0 25 0 0 |
0 30 0 0 25 0 0 |
10 26 0 0 32 0 0 |
0 31 1 0 32 0 0 |
0 31 0 0 27 0 0 |
0 33 0 0 27 0 0 |
0 27 0 0 25 0 0 |
Total |
134 |
153 |
158 |
153 |
159 |
159 |
154 |
147 |
157 |
140 |
Mean SD CV |
151.40 8.53 5.63 |
|||||||||
CD |
- |
|||||||||
No. of days Juvenile per day |
15 8.93 |
15 10.20 |
15 10.53 |
15 10.20 |
15 10.60 |
15 10.60 |
15 10.27 |
15 9.80 |
15 10.47 |
15 9.33 |
Mean SD CV |
10.09 0.57 5.63 |
|||||||||
CD |
- |
|||||||||
Remark: SD = Standard deviation
CV = Coefficient of variance (%)
CD = Deviation from the mean of control (Reduction %)
The Number of Juveniles at 100.0 mg/L nominal loading rate WAF
Day |
Replicate |
|||||||||
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
|
1 2 3 4 5 6 7 |
0 0 0 0 0 0 11 |
0 0 0 0 0 0 13 |
0 0 0 0 0 0 15 |
0 0 0 0 0 0 13 |
0 0 0 0 0 0 14 |
0 0 0 0 0 0 9 |
0 0 0 0 0 0 9 |
0 0 0 0 0 0 10 |
0 0 0 0 0 0 13 |
0 0 0 0 0 0 15 |
8 9 10 11 12 13 14 |
0 0 25 0 1 32 0 |
0 0 27 0 0 31 0 |
0 0 27 0 0 33 0 |
0 0 23 0 0 28 1 |
0 0 26 0 0 31 0 |
0 0 26 0 0 29 0 |
0 0 28 0 0 39 0 |
0 0 25 0 0 41 0 |
0 0 26 0 0 32 0 |
0 0 25 0 0 28 0 |
15 16 17 18 19 20 21 |
0 32 0 0 22 0 0 |
0 31 0 0 2 29 0 |
0 33 0 0 2 32 0 |
0 36 0 0 1 34 0 |
0 34 0 M - - - - |
0 31 0 0 35 0 0 |
0 32 0 0 31 0 0 |
0 41 0 0 32 0 0 |
0 34 0 0 36 0 0 |
0 37 0 0 32 0 0 |
Total |
123 |
133 |
142 |
136 |
E |
130 |
139 |
149 |
141 |
137 |
Mean SD CV |
136.67 7.50 5.49 |
|||||||||
CD |
9.7 |
|||||||||
No. of days Juvenile per day |
15 8.20 |
15 8.87 |
15 9.47 |
15 9.07 |
E E |
15 8.67 |
15 9.27 |
15 9.93 |
15 9.40 |
15 9.13 |
Mean SD CV |
9.11 0.50 5.49 |
|||||||||
CD |
9.7 |
|||||||||
Remark: SD = Standard deviation
CV = Coefficient of variance (%)
CD = Deviation from the mean of control (Reduction %)
Appearance of the first brood at Control
|
Replicate |
|||||||||
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
|
First brood (day) |
7 |
7 |
7 |
7 |
7 |
7 |
7 |
7 |
7 |
7 |
Mean SD CV |
7.00 0.00 0.00 |
|||||||||
CD |
- |
|||||||||
Remark: SD = Standard deviation
CV = Coefficient of variance (%)
CD = Deviation from the mean of control (Reduction %)
Appearance of the first brood at 100.0 mg/L nominal loading rate WAF
|
Replicate |
|||||||||
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
|
First brood (day) |
7 |
7 |
7 |
7 |
7 |
7 |
7 |
7 |
7 |
7 |
Mean SD CV |
7.00 0.00 0.00 |
|||||||||
CD |
0.0 |
|||||||||
Remark: SD = Standard deviation
CV = Coefficient of variance (%)
CD = Deviation from the mean of control (Reduction %)
Body length of the parent animals at the end of the test at Control
|
Replicate |
|||||||||
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
|
Body length (mm) |
4.0 |
4.2 |
4.5 |
4.3 |
4.6 |
4.4 |
4.5 |
4.5 |
4.1 |
4.3 |
Mean SD CV |
4.34 0.20 4.50 |
|||||||||
CD |
- |
|||||||||
Remark:SD = Standard deviation
CV = Coefficient of variance (%)
CD = Deviation from the mean of control (Reduction %)
Body length of the parent animals at the end of the test at 100.0 mg/L nominal loading rate WAF
|
Replicate |
|||||||||
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
|
Body length (mm) |
4.0 |
4.1 |
4.0 |
4.2 |
E |
4.3 |
4.2 |
4.4 |
3.9 |
3.8 |
Mean SD CV |
4.10 0.19 4.72 |
|||||||||
CD |
5.5 |
|||||||||
Remark: SD = Standard deviation
CV = Coefficient of variance (%)
CD = Deviation from the mean of control (Reduction %)
Mortality and reproductive potency during the range-finding test
LOWINOX® CPL WAF prepared at the given loading rate (mg/l) |
Number Daphnia exposed |
Cumulative number of dead/immobile parental daphnids |
||||||
Day 1 |
Day 2 |
Day 3 |
Day 4 |
Day 5 |
Day 6 |
Day 7 |
||
Control |
5 5 |
0 (0) 0 (0) |
0 (0) 0 (0) |
0 (0) 0 (0) |
0 (0) 0 (0) |
0 (1) 0 (0) |
0 (5) 0 (4) |
0 (5) 0 (4) |
1.0 |
5 5 |
0 (0) 0 (0) |
0 (0) 0 (0) |
0 (0) 0 (0) |
0 (0) 0 (0) |
0 (0) 0 (0) |
0 (2) 0 (0) |
0 (5) 0 (5) |
10 |
5 5 |
0 (0) 01(0) |
0 (0) 0 (0) |
0 (0) 0 (0) |
0 (0) 0 (0) |
0 (1) 0 (1) |
0 (1) 0 (3) |
0 (5) 0 (5) |
100 |
5 5 |
01(0) 01(0) |
01(0) 01(0) |
0 (0) 0 (0) |
0 (0) 0 (0) |
0 (0) 0 (0) |
0 (0) 0 (1) |
0 (5) 0 (5) |
( ) Number of parents with eggs in their brood pouch
1Some of the daphnids were observed to be trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.
Mortality (immobility) of parental daphnids at the end of the test
LOWINOX® CPL WAF prepared at the given loading rate (mg/l) |
Introduced |
Mobile |
Immobile |
%Immobility |
Control |
20 |
20 |
0 |
0 |
1.0 |
20 |
16 |
4 |
20 |
Group mean cumulative number of juveniles per surviving parent and reduction of reproduction at the end of the test
Parameter |
LOWINOX® CPL, WAF prepared at the given loading rate (mg/l) |
|
Control |
1.0 |
|
Mean Std.Dev. n CV % Reproduction |
91 15 20 16 - |
72 30 16 41 21* |
- Not applicable
* Statistically significant (p<0.05)
Incidence of immobilised offspring and aborted eggs
Abnormality |
LOWINOX® CPL, WAF prepared at the given loading rate (mg/l) |
|
Control |
1.0 |
|
Immobile offspring Aborted eggs |
136 - |
154 - |
-Not observed
Group mean body lengths (mm) and reduction of growth of parental daphnids at the end of the test
LOWINOX® CPL concentration (mg/l) |
Mean (mm) |
Std. Dev. |
n |
%Reduction |
Control 1.0 |
4.02 3.90 |
0.11 0.19 |
20 16 |
- 3# |
#Statistically significant (p<0.05) but biologically insignificant (<10%)
Description of key information
21 -day NOEC for reproductive effect was determined to be 100.0 mg/L nominal loading rate and the LOEC was determined to be as higher than this tested level.
Key value for chemical safety assessment
Fresh water invertebrates
Fresh water invertebrates
- Effect concentration:
- 100 mg/L
Additional information
Daphnia magna were exposed in a semi-static test to saturated aqueous test media containing the test item at 100.0 mg/L nominal loading rate WAF in a limit test for 21 days.
The 21-days reproduction test was carried out in a semi-static renewal system, with a renewal frequency of 24 hours based on analytical method validation (Study Code: 16/451-316AN) (due to that test item is a UVCB substance with very poor solubility).
Concentration of the test item in the test solution was analysed by the validated method - when freshly prepared and at renewal - on one occasion during the first week then these determinations were repeated weekly intervals thereafter based on the performed analytical method validation. Fully defined Elendt M4 medium was used as dilution water for the test solution.
Test item concentration could not be detected and therefore measured concentration was below the Limit of Detection (LOD = 0.75 μg/mL) for all WAF samples (freshly prepared and at renewal) during the experiment. The biological results are based on the nominal loading rate WAF. The maximum amount of test material in the medium has been exposed applying this procedure.
Test item exposure and control group comprised 10 Daphnids. The test animals were maintained individually, one per test vessel, with ~80 mL of test solution in each vessel.
The average cumulative number of young per parent animal in the control was 151.40 with a variation coefficient of 5.63 %.
Only one dead animal was observed in the test item treated group of 100.0 mg/L nominal loading rate WAF during the whole experiment. Mortality was not observed in the control group.
The reproduction of the surviving parent animals was not significantly different from the control group in the test item treated group of 100.0 mg/L nominal loading rate WAF (2 Sample t-Test, α = 0.05).
Statistically significant difference was not observed concerning the body length of the surviving parent animals either (2 Sample t-Test, α = 0.05).
The time to production of first brood was the same in the test item group of 100.0 mg/L WAF as in the control group during the test.
In the 21-days Reproduction test on Daphnia magna with LOWINOX® CPL the 21-day LC50-value of parents and the 21-day reproduction EC10, EC20 and EC50 values were determined to be higher than 100.0 mg /L nominal loading rate WAF (considered to be a saturated solution). The NOEC for reproductive effect was determined to be 100.0 mg/L nominal loading rate and the LOEC was determined to be as higher than this tested level.
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