Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Currently viewing:

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From 2000-04-04 to 2000-06-21
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
acceptable restriction was that analytical measurements on the test material were not conducted.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
Principles of method if other than guideline:
not applicable.
GLP compliance:
yes (incl. QA statement)
Remarks:
2000-04-26
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
none.
Analytical monitoring:
no
Details on sampling:
- Concentrations: 1.5, 4.8, 15, 48, and 150 mg/L
- Sampling method: The concentration and stability of the test material in the test solutions were verified by chemical analysis on days 0 (fresh media), 2, 5, 7, 9, 12, 14, 16, 19 and 21 (old media)
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Loading rates
Based on the results of an acute toxicity test to 1st instar Daphnia magna (Safepharm Laboratories Project Number 525/259) the following nominal loading rates were assigned to the study: 1.5, 4.8, 15, 48 and 150 mg/l.
- Method:A WAF of nominal loading rate of 1000 mg/L was prepared, in duplicate, in reconstituted water. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1 hour standing period each of the mixtures was filtered through 0.2 µm filters and the concentration of the test material in the filtered WAFs verified by Dissolved Organic Carbon (DOC) analysis. Filtration was conducted as undissolved test material was observed throughout the water columns of the test loading rates at the end of each preparation period during WAF method development work. It was considered that careful siphoning of the loading rates would not have removed all of the undissolved test material as it may have exhibited a physical effect on the test organisms during testing.
- Eluate: Reconstituted water.
- Differential loading: Amounts of test material (15, 48, 150, 480, 1500 mg) were each separately added to the vortex created in 10 L of reconstituted water to give the 1.5, 4.8, 15, 48, and 150 mg/L loading rates.
- Controls: The control group was maintained under identical conditions but not exposed to the test material.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnids
- Strain/clone: 1st instar
- Source: in-house laboratory
- Age of parental stock (mean and range, SD): no data
- Feeding during test
- Food type: Chlorella sp.
- Amount: 30 x 10^9 cells per day
- Frequency: daily

ACCLIMATION
- Acclimation period: no data
- Acclimation conditions (same as test or not): Adults were maintained in polypropylene vessels containing approximately 2 L of reconstituted water at 21°C.
- Type and amount of food: Chlorella sp. 30 x 10^9 cells per day
- Feeding frequency: daily

On a daily basis, the numbers of live and dead of the parental generation, the numbers of live and dead filial Daphnia, and the number of discarded unhatched eggs were counted. An assessment was also made of the general condition and size of the parental Daphnia as compared with the controls.
The number of Daphnia with eggs or young in the brood pouch was determined at each meadia renewal. Young daphnids were considered to be dead if no sign of movement was apparent during microscopic examination. Adult Daphnia which were unable to swim for approximately 15 sec after gentle agitation, were considered to be dead. An immobilisation criterion for the young daphnids was considered to be inappropriate due to the large numbers of off-spring produced in the flasks.
At the end of the study, the length of each surviving parent animal was determined.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
none.
Hardness:
The water hardness was observed to be in the range 218 to 256 mg/l as CaCO3 in the control and the highest surviving test group throughout the study.
Test temperature:
21.0°C
pH:
7.7-8.0
Dissolved oxygen:
8.0-8.4 mg O2/L
Salinity:
not applicable.
Nominal and measured concentrations:
nominal concentrations: 0, 1.5, 4.8, 15, 48, 150 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: glass flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 150 mL
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): 3 times per week (days 2, 5, 7, 9, 12, 14, 16 and 19).
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: 16 light:8 dark
- Light intensity: ca. 132 - 152 lux.

Reference substance (positive control):
no
Duration:
14 d
Dose descriptor:
EL50
Effect conc.:
48 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Remarks on result:
other: 33-69
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
27 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Remarks on result:
other: 15-48
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
31 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 27-37
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
48 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
15 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Details on results:
none
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
none.

Study data are presented in attached document (cf. Tables.pdf)

Validity criteria fulfilled:
yes
Conclusions:
The 14 and 21 -day EL50 (immobilisation) values, based on the nominal loading rates, for the parental Daphnia generation were calculated to be 48 and 27 mg/L loading rate filtered WAF with 95% confidence limits of 33 -69 mg/L loading rate filtered WAF and 15-48 mg/L loading rate filtered WAF respectively. The 21 -day EL50 (reproduction) was calculated to be 31 mg/L loading rate filtered WAF with 95% confidence limits of 37-37 mg/L. The LOEC is considered to be 48 mg/L loading rate filtered WAF on the basis that at this loading there were significant mortalities (immobilisation) observed in the parental generation. The NOEC is considered to be 15 mg/L loading rate filtered WAF on the basis that at this loading rate there were no mortalities (immobilisation) observed in the parental generation and that there were no significant differences between the control and the 15 mg/L loading rate filtered WAF test group in terms of the number of live young produced per adult by day 21.
Executive summary:

Guideline: A study was performed to assess the effect of the test material on the reproduction of Daphnia magna over a 21 day period. The method followed that described in the OECD Guidelines No 211 "Daphnia magna, Reproduction test".

Methods: Based on the results of an acute toxicity study, Daphnia magna were exposed (10 replicates of a single daphnid per group) to filtered WAF of the test material over a range of nominal loading rates of 1.5, 4.8, 15, 48, 150 mg/L for a period of 21 days. The test solutions were renewed 3 times per week. The numbers of live and dead adult Daphnia and young daphnids were determined daily. The Daphnia were fed daily with an algal suspension.

Results: Analysis of the test loading rates at 0 hours showed the measured concentrations to range from 0.106 to 0.826 mg/L whilst analysis of the old or expired media sampled throughout the study showed measured concentrations to range from less than the limit of quantification of the analytical method to 0.863 mg/L. These results were in-line with the recovery and stability analyses performed on the WAFs which showed the test material to be unstable in both light and dark conditions at the lower test concentrations. On the majority of sampling occasions increasing measured concentrations were shown with increasing loading rates.

Given that the toxicity cannot be attributed to any one or more of the components in the WAF, but to the mixture as a whole, the results are based on nominal loading rates only.

The 14 and 21 -day EL50 (immobilisation) values, based on the nominal loading rates, for the parental Daphnia generation were calculated to be 48 and 27 mg/L loading rate filtered WAF with 95% confidence limits of 33 -69 mg/L loading rate filtered WAF and 15 -48 mg/L loading rate filtered WAF respectively.

The 21 -day EL50 (reproduction) was calculated to be 31 mg/L loading rate filtered WAF with 95% confidence limits of 37 -37 mg/L.

The LOEC is considered to be 48 mg/L loading rate filtered WAF on the basis that at this loading there were significant mortalities (immobilisation) observed in the parental generation.

The NOEC is considered to be 15 mg/L loading rate filtered WAF on the basis that at this loading rate there were no mortalities (immobilisation) observed in the parental generation and that there were no significant differences between the control and the 15 mg/L loading rate filtered WAF test group in terms of the number of live young produced per adult by day 21.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 July 2020 to 15 January 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
2012
Deviations:
yes
Remarks:
with no impact on results or integrity of the study (see below)
Qualifier:
according to guideline
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Version / remarks:
Commission Regulation (EC) No. 440/2008
Deviations:
yes
Remarks:
with no impact on results or integrity of the study (see below)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Range-finding test: A sample of each test concentration was taken for chemical analysis on Days 0 and 3 in order to determine the stability of the test item under test conditions. All samples were analyzed on the day of sampling. Duplicate samples were taken and stored frozen.
- Definitive test: Water samples were taken from the control and each surviving test group (replicates pooled) for quantitative analysis. Samples of the fresh test preparations were taken on Days 0, 5, 12 and 19 and of the expired test preparations on Days 2, 7, 14 and 21. Samples taken on Days 0, 2, 7, 12, 19 and 21 were analyzed on the day of sampling whereas the samples taken on Day 5 and 14 were stored frozen prior to analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
TEST WATER
- The reconstituted water (ISO medium) used for the range finding and definitive tests was the same as that used to maintain the stock animals and is defined below.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST SYSTEM
- The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
- Adult Daphnia were maintained in 150 mL glass vessels containing ISO medium (see below) in a temperature controlled room at approximately 20 C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Raphidocelis subcapitata (formally known as Pseudokirchneriella subcapitata)) and fish food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

TEST ORGANISM OBSERVATIONS
- On a daily basis the numbers of live and dead of the "Parental" (P1) generation, the numbers of live and dead "Filial" (F1) Daphnia and the number of discarded unhatched eggs were counted. An assessment was also made of the general condition and size of the parental Daphnia as compared with the controls.
- Young daphnids were considered to be dead if no sign of movement was apparent during microscopic examination. Adult Daphnia which were unable to swim for approximately 15 seconds after gentle agitation (ie. immobile), were considered to be dead. An immobilization criterion for the young daphnids was considered to be inappropriate due to the large numbers of off-spring produced in the flasks.
- At the end of the test, the length of each surviving parent animal was determined.

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
Not applicable
Hardness:
244 to 270 mg/L as CaCO3
Test temperature:
19 °C to 21 °C
pH:
7.7 to 8.4 (control group)
Dissolved oxygen:
≥ 8.4 mg O2/L
Salinity:
Not applicable
Conductivity:
Not reported
Nominal and measured concentrations:
- Range-finding test: 1.0, 10 and 100 mg/L (nominal test item loading rates)
- Definitive test: 4.5, 10, 22, 45 and 100 mg/L (nominal test item loading rates|)
Details on test conditions:
PURPOSE OF STUDY
- Daphnia magna is a freshwater invertebrate representative of a wide variety of natural habitats, and can therefore be considered as an important non-target organism in freshwater ecosystems.
- In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. In cases where the test item is a complex mixture and is poorly soluble in water, an approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2019 and Singer et al 2000), is to expose organisms to a WAF of the test item. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. At the completion of mixing and following a settlement period, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.

EXPERIMENTAL DESIGN AND STUDY CONDUCT
- Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a WAF of the test item.

VALIDATION OF MIXING PERIOD
- Preliminary work was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF.

RANGE-FINDING TEST
- The test concentrations to be used in the definitive test were determined by a preliminary range finding test.
- In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/L.
- Nominal amounts of test item (10, 20 and 200 mg) were each separately added to the surface of 10, 2 and 2 L of test water to give the 1.0, 10 and 100 mg/L loading rates respectively. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 3 hours and the mixtures allowed to stand for 1 hour. A control WAF was prepared under identical conditions but not exposed to the test item. Visual observations made on the test WAFs indicated showed no microdispersions were observed; however, the WAFs was filtered through a glass wool plug (2 to 4 cm in length) as a precautionary measure. A 10 mL and a 1.0 mL pipette, covered at one end with parafilm, were submerged within the vessels (sealed end down to mid depth within the vessel). A length of Tygon tubing was attached to the 10 mL pipette and the parafilm was removed using the 1.0 mL pipette. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid depth siphoning (the first approxiamte 100 mL discarded). Microscopic observations of the WAFs were performed after filtering and showed no micro dispersions of test item to be present.
- In the range finding test, for each concentration a single daphnid was placed in 100 mL of the test preparation in 150 mL glass vessels which were then covered with a plastic lid to reduce evaporation. For each test and control group five replicate test vessels were prepared. The water temperature was maintained at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light and 8 hours darkness with 20-minute dawn and dusk transition periods for 10 days.
- The control group was maintained under identical conditions but not exposed to the test item.
- The test preparations were renewed on Days 3, 5 and 7. The adult Daphnia were transferred to fresh media by wide-bore pipette before the contents of each vessel were passed through a fine mesh. Young daphnids (live and dead) and any unhatched eggs were collected on the mesh and counted using a stereo microscope before being discarded.
- Each daphnid received approximately 5 to 20 µL of an algal suspension (Raphidocelis subcapitata) daily, supplemented by the addition of approximately 20 µL of fish food suspension on Days 0, 1, 2 and 3. Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.

INITIAL TEST
- An initial test was conducted following the same method as for the definitive test; however, this test was terminated on Day 5 as a result of significant sub-lethal effects in all test groups. As the observations did not match the observations in the range-finding test and were affecting all test groups similarly, the test was restarted using the same range of concentrations.

DEFINITIVE TEST
- Based on the results of a preliminary range finding test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to WAFs of the test item over a range of test concentrations of 4.5, 10, 22, 45 and 100 mg/L for a period of 21 days. The test solutions were renewed three times per week throughout the test.
- Nominal amounts of test item (9.0, 20, 44, 90 and 200 mg) were each separately added to the surface of 2 L of test water to give the 4.5, 10, 22, 45 and 100 mg/L loading rates respectively. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 3 hours and the mixtures allowed to stand for 1 hour. Microscopic inspection of the WAFs showed no micro dispersions or undissolved test item to be present. To remove the aqueous phase or WAF, a 10 mL and a 1.0 or 2.0 mL pipette, covered at one end with parafilm, were submerged within the vessels (sealed end down to mid depth within the vessel). A length of Tygon tubing was attached to the 10 mL pipette and the parafilm was removed using the 2.0 mL pipette. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid depth siphoning (the first 75 to 100 mL discarded; however no record of the volume discarded was recorded for the media change on Day 5) to give the 4.5, 10, 22, 45 and 100 mg/L loading rate WAFs.

EXPOSURE CONDITIONS
- For each concentration a single daphnid was placed in 100 mL of the test preparation in 150 mL glass vessels which were then covered with a Perspex cover to reduce evaporation. For each test and control group 10 replicate test vessels were prepared. The test vessels were maintained in a temperature controlled room at 18 to 22 C with a maximum deviation of ± 1 °C with a photoperiod of 16 hours light (not exceeding 1500 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods for 21 days. The test vessels were not aerated. The diluent water only was aerated prior to use.
- The control group was maintained under identical conditions but not exposed to the test item.
- The test preparations were renewed three times per week on Days 2, 5, 7, 9, 12, 14, 16 and 19. The adult Daphnia were transferred to fresh media by wide bore pipette before the contents of each vessel were passed through a fine mesh. Young daphnids (live and dead) and any unhatched eggs were collected on the mesh and counted before being discarded.
- Each daphnid received approximately 5 to 20 µL of an algal suspension (Raphidocelis subcapitata) daily, supplemented by the addition of approximately 20 µL of fish food suspension on Days 0, 1, 2 and 3. Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.

WATER QUALITY CRITERIA
- Dissolved oxygen concentrations, pH and temperature were recorded before and after each test media renewal.
- The pH and dissolved oxygen concentration were measured using a Hach HQ30d Flexi handheld meter whilst the temperature was measured using a digital thermometer. Measurements were made on one replicate for each test concentration.
- The water hardness of the control and the highest surviving test concentration in the fresh and old media was measured once per week.

VALIDATION CRITERIA
- For the test to be valid, the following criteria should be fulfilled:
(i) Mortality of parent test animals in the controls should not exceed 20 % at the end of the test.
(ii) The mean number of living offspring in the control should be at least 60 per surviving adult Daphnia at the end of the test.
(iii) The coefficient of variation around the mean number of living offspring produced per parent animal in the control should be  25 %.
(iv) No ephippia are produced in the controls.
(v) Dissolved oxygen concentration should remain > 3 mg O2/L throughout the test.
(vi) pH of the controls should be within the range 6 to 9 pH units and not vary by > 1.5 units throughout the test.

MAJOR COMPUTERIZED SYSTEMS
- Building management: Delta control system
- Statistics: RoxRat
- Electronic communication system: Veeva QMS
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
EL10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
other: EL20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
EL10
Effect conc.:
28 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
reproduction
Remarks on result:
other: 95 % confidence limits 7.4 to 107 mg/L koading rate WAF
Duration:
21 d
Dose descriptor:
other: EL20
Effect conc.:
52 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
reproduction
Remarks on result:
other: 95 % confidence limits 12 to 223 mg/L loading rate WAF
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
45 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EL10
Effect conc.:
71 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
other: body length
Remarks on result:
other: 95 % confidence limits 33 to 151 mg/L loading rate WAF
Duration:
21 d
Dose descriptor:
other: EL20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
other: body length
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
other: body length
Key result
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
45 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
other: body length
Duration:
21 d
Dose descriptor:
LOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
other: body length
Details on results:
RANGE-FINDING TEST
- Cumulative immobilization and sub lethal effects data from the exposure of Daphnia magna to the test item during the range finding test were given in Table 1 of the full study report.
- No immobilization was observed at the test concentrations of 1.0 and 10 mg/L, however, 20 % immobilization was observed at 100 mg/L.
- No sub lethal effects of exposure were observed in any of the parental generation Daphnia.
- No reduction of reproduction in the first brood was observed at a test concentration of 10 mg/L; however, in the concentrations at 1.0 and 100 mg/L the numbers of neonates observed in the first brood was lower than in the control. The reduction in neonates in the 1.0 mg/L test group did not follow a dose response and was therefore considered not necessarily to be as a result of exposure to the test item.
- Chemical analysis of the fresh test preparations on Day 0 showed the measured boron concentration (corrected for boron concentration in the control media) in the 100 mg/L loading rate was 0.87 mg/L, corresponding to a test item concentration of 73 mg/L. Chemical analysis of the old test preparations on Day 3 showed the corrected measured boron concentration in the 100 mg/L loading rate was 0.74 mg boron/L, corresponding to a test item concentration of 63 mg/L. Measured concentrations from the 1.0 and 10 mg/L loading rate were below the limit of quantification.

DEFINITIVE TEST
- Based on the results of a preliminary range finding test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to a WAFs of the test item over a range of test concentrations of 4.5, 10, 22, 45 and 100 mg/L for a period of 21 days. The test solutions were renewed three times per week throughout the test.

VERIFICATION OF TEST CONCENTRATIONS
- Chemical analysis of the fresh test preparations on Days 0, 5, 12 and 19 showed measured boron concentrations (corrected for boron concentrations in the control media) were between 0.037 and 1.2 mg boron/L, corresponding to test item concentrations of 3.1 and 99 mg/L. Chemical analysis of the old test preparations on Days 2, 7, 14 and 21 showed corrected measured boron concentrations were between 0.026 and 1.2 mg boron/L, corresponding to test item concentrations of 2.2 and 99 mg/L.
- Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.
TEST ORGANISM OBSERVATIONS
- The observations for the control and each test group were summarized in Table 2 of the full study report.
- The total cumulative production of live young excluding accidental and inadvertent mortalities was given in Table 3 of the full study report.
- The number of live young produced per adult excluding accidental and inadvertent mortalities was shown in Table 4 of the full study report.
- The total number of offspring per parent Daphnia (for each replicate) alive at the end of the test excluding accidental and inadvertent mortalities was shown in Figure 1 of the full study report.
- Data for each individual replicate vessel were given in Appendix 2 to Appendix 7 of the full study report.

VALIDATION CRITERIA
- The validation criteria achieved during the test are summarized in the table below.

LETHAL EFFECTS ON THE PARENTAL GENERATION (P1)
- Low level mortality (immobilization) was observed in the control and all test loading rates, however, statistical analysis of the mortality data using the Multiple Sequentially-rejective Fisher Test after Bonferroni-Holm showed that the observed mortalities were not significantly different (P  0.05) when compared to the control group.

SUB-LETHAL EFFECTS ON THE PARENTAL GENERATION (P1)
- The body length of surviving adults at Day 21 was given in Table 5 of the full study report.
- The relationship between body length and concentration at Day 21 was given in Figure 2 of the full study report.
- After 21 days there were no statistically significant differences (P ≥ 0.05) between the control, 4.5, 10, 22 and 45 mg/L loading rate WAF groups in terms of length of the daphnids after 21 days exposure to the test item; however a significant difference (P < 0.05) was determined between the control and the 100 mg/L loading rate WAF group.
- Other sub-lethal observations of the parental generation Daphnia were as follows; in the control, one daphnia coated with algae and one daphnia was pale; however, parental Daphnia in all test groups were recorded as small, pale or small and pale until Day 9 for the 4.5, 10, 22 and 45 mg/L loading rate WAF groups and until Day 11 for the 100 mg/L loading rate WAF group.

EFFECTS ON REPRODUCTION
- The relationship between the number of live young produced per adult after 21 days and concentration at Day 21 was given in Figure 3 of the full study report.
- After 21 days there were no statistically significant differences (P ≥ 0.05) between the control, 4.5, 10, 22 and 45 mg/L loading rate WAF groups in terms of the number of live young produced per adult after 21 days exposure to the test item; however a significant difference (P < 0.05) was determined between the control and the 100 mg/L loading rate WAF group

EFFECTS ON THE FILIAL GENERATION (F1)
- Information on the effects of the test item on the F1 generation is limited, since, by study design, the young are removed soon after liberation from the brood pouch, however, an assessment made at each media renewal showed in the control group five broods of the "filial" daphnids produced were smaller than normal, in the 4.5 mg/L loading rate WAF group two broods were pale and two broods were smaller than normal, in the 10 mg/L loading rate WAF group one brood was pale and three broods were trapped at the surface whereas all broods produced in the 22, 45 and 100 mg/L loading rate WAF groups were normal in appearance.
- Young were first produced in the control test group on Day 8 of the test.
- There were no unhatched eggs or dead young in the control or any of the treatment groups.

WATER QUALITY CRITERIA
- The results of the water quality measurements were given in Table 6 of the full study report.
- Temperature was maintained at 19 C to 21 C throughout the test, while there were no treatment related differences for oxygen concentration or pH.
- The results of the water hardness measurements were given in Table 7 of the full study report. The water hardness was observed to be in the range 244 to 270 mg/L as CaCO3 in the control and the highest surviving test group throughout the test.
- Light intensity measurements were reported in Table 8 of the full study report. Throughout the test the light intensity was observed to be in the range 722 to 912 lux.
VORTEX DEPTH MEASUREMENTS
- The vortex depth was recorded at the start and end of the mixing period and was observed to have formed a dimple at the media surface.

OBSERVATIONS ON TEST ITEM SOLUBILITY
- Observations on the test media were carried out during the mixing and testing of the WAFs.
- At the start of the mixing period all loading rates were observed to be a clear colorless water column with test item floating at the surface. After 3 hours stirring and a 1 Hour standing period all loading rates were observed to remain as at the start of stirring. Microscopic inspection of the WAF showed no micro dispersions or undissolved test item to be present. After siphoning and for the duration of the test, all loading rates were observed to be clear, colorless solutions.
Reported statistics and error estimates:
- Outlier test: One of the control replicates produced considerably more neonates than any of the other replicates which resulted in the control group failing the validity criteria for the coefficient of variation for neonates produced. Therefore prior to the statistical analysis, the number of neonates produced in the control replicates were analyzed using the Dixon and Hartley Outlier Test using the ToxRat computer software package (ToxRat Solutions GmbH).
- ELx calculations: No statistically significant concentration responses were determined for immobilization; however, ELx values were determined by Probit analysis using Linear Maximum-Likelihood regression. The ELx (reproduction) values and associated confidence limits after 21 days were determined by 3-Parameter Normal Cumulative Distribution Function. The ELx (body length) values and associated confidence limits after 21 days were determined by the 3-Parameter Normal Cumulative Distribution Function. All results were calculated using the ToxRat computer software package (ToxRat Solutions GmbH).
- LOEL and NOEL determinations: For the estimation of the Lowest Observed Effect Loading Rate (LOEL) and the No Observed Effect Loading Rate (NOEL) the immobilization data for the control and each test group were compared using the Multiple Sequentially-rejective Fisher Test after Bonferroni Correction. The numbers of live young produced per adult over the duration of the test data for the control and each test group obtained on termination of the test were compared using Williams Multiple Sequential t-test. Daphnia length data, determined for the surviving daphnids on termination of the test, were compared using the Multiple Sequentially -rejective Welsh-t-test after Bonferroni-Holm. All results were calculated using the ToxRat computer software package (ToxRat Solutions GmbH).

VALIDATION CRITERIA

Criterion

Required

Actual

Control mortality

≤ 20 %

20 %

Mean number of live young per surviving adult (control group)

≥ 60 after 21 days

89.9 *

Coefficient of variation for control group**

≤ 25 %

16.1 % *

No ephippia produced

0

0

Dissolved oxygen

> 3 mg O2/L

≥ 8.4 mg O2/L

pH (control group)

6 to 9 (variation ≤ 1.5)

7.7 to 8.4 (variation 0.7)

*

Excluding data from Control replicate 7 which was determined to be a statistically significant outlier

**

Based on total number of living offspring per parent animal alive at the end of the test

Validity criteria fulfilled:
yes
Conclusions:
The 21-day nominal No Observed Effect Loading Rate (NOELR) reported in an investigation of Daphnia magna was 45 mg/L based on reproduction and body length.
Executive summary:

GUIDELINE

The study was performed to assess the chronic toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2012) No 211, "Daphnia magna Reproduction Test" referenced as Method C.20 of Commission Regulation (EC) No. 440/2008.

 

METHODS

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as Water Accommodated Fractions (WAFs). Based on the results of a preliminary range‑finding test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to a WAF of the test item over a range of nominal loading rates of 4.5, 10, 22, 45 and 100 mg/L for a period of 21 days. The test solutions were renewed three times per week throughout the test. The numbers of live and dead adult Daphnia and young daphnids (live and dead) were determined daily. The Daphnia were fed daily with algal suspension, supplemented with fish food suspension on Days 0, 1, 2 and 3.

 

RESULTS

Chemical analysis of the fresh test preparations on Days 0, 5, 12 and 19 showed measured boron concentrations (corrected for boron concentrations in the control media) were between 0.037 and 1.2 mg boron/L, corresponding to test item concentrations of 3.1 and 99 mg/L. Chemical analysis of the old test preparations on Days 2, 7, 14 and 21 showed corrected measured boron concentrations were between 0.026 and 1.2 mg boron/L, corresponding to test item concentrations of 2.2 and 99 mg/L.

 

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

 

Exposure of Daphnia magna to the test item gave the following results based on the nominal loading rates:

Parameter

Endpoint

Concentration (mg/L loading rate WAF)

Immobilisation

EL10

> 100

Immobilisation

EL20

> 100

Immobilisation

EL50

> 100

Immobilisation

No Observed Effect Loading Rate

100

Immobilisation

Lowest Observed Effect Loading Rate

Not determined

Reproduction

EL10

28

(95 % confidence limits 7.4 to 107)

Reproduction

EL20

52

(95 % confidence limits 12 to 223)

Reproduction

EL50

> 100

Reproduction

No Observed Effect Loading Rate

45

Reproduction

Lowest Observed Effect Loading Rate

100

Body length

EL10

71

(95 % confidence limits 33 to 151)

Body length

EL20

> 100

Body length

EL50

> 100

Body length

No Observed Effect Loading Rate

45

Body length

Lowest Observed Effect Loading Rate

100

Description of key information

Key study

The 21-day nominal No Observed Effect Loading Rate (NOELR) reported in an investigation of Daphnia magna was 45 mg/L based on reproduction and body length (OECD 211 and EU Method C.20).

Supporting study

The 14 and 21-day EL50 (immobilisation) values, based on the nominal loading rates, for the parental Daphnia generation were calculated to be 48 and 27 mg/L loading rate filtered WAF with 95 % confidence limits of 33 to 69 mg/L loading rate filtered WAF and 15 to 48 mg/L loading rate filtered WAF respectively. The 21-day EL50 (reproduction) was calculated to be 31 mg/L loading rate filtered WAF with 95 % confidence limits of 37 to 37 mg/L. The LOEC is considered to be 48 mg/L loading rate filtered WAF on the basis that at this loading there were significant mortalities (immobilisation) observed in the parental generation. The NOEC is considered to be 15 mg/L loading rate filtered WAF on the basis that at this loading rate there were no mortalities (immobilisation) observed in the parental generation and that there were no significant differences between the control and the 15 mg/L loading rate filtered WAF test group in terms of the number of live young produced per adult by day 21 (OECD 211).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
NOEC
Effect concentration:
45 mg/L

Additional information

Key study

The key study was performed to assess the chronic toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2012) No 211, "Daphnia magna Reproduction Test" referenced as Method C.20 of Commission Regulation (EC) No. 440/2008.

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as Water Accommodated Fractions (WAFs). Based on the results of a preliminary range‑finding test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to a WAF of the test item over a range of nominal loading rates of 4.5, 10, 22, 45 and 100 mg/L for a period of 21 days. The test solutions were renewed three times per week throughout the test. The numbers of live and dead adult Daphnia and young daphnids (live and dead) were determined daily. The Daphnia were fed daily with algal suspension, supplemented with fish food suspension on Days 0, 1, 2 and 3.

Chemical analysis of the fresh test preparations on Days 0, 5, 12 and 19 showed measured boron concentrations (corrected for boron concentrations in the control media) were between 0.037 and 1.2 mg boron/L, corresponding to test item concentrations of 3.1 and 99 mg/L. Chemical analysis of the old test preparations on Days 2, 7, 14 and 21 showed corrected measured boron concentrations were between 0.026 and 1.2 mg boron/L, corresponding to test item concentrations of 2.2 and 99 mg/L.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

Exposure of Daphnia magna to the test item gave the following results based on the nominal loading rates:

The key study was performed to assess the chronic toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2012) No 211, "Daphnia magna Reproduction Test" referenced as Method C.20 of Commission Regulation (EC) No. 440/2008.

 

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as Water Accommodated Fractions (WAFs). Based on the results of a preliminary range‑finding test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to a WAF of the test item over a range of nominal loading rates of 4.5, 10, 22, 45 and 100 mg/L for a period of 21 days. The test solutions were renewed three times per week throughout the test. The numbers of live and dead adult Daphnia and young daphnids (live and dead) were determined daily. The Daphnia were fed daily with algal suspension, supplemented with fish food suspension on Days 0, 1, 2 and 3.

 

Chemical analysis of the fresh test preparations on Days 0, 5, 12 and 19 showed measured boron concentrations (corrected for boron concentrations in the control media) were between 0.037 and 1.2 mg boron/L, corresponding to test item concentrations of 3.1 and 99 mg/L. Chemical analysis of the old test preparations on Days 2, 7, 14 and 21 showed corrected measured boron concentrations were between 0.026 and 1.2 mg boron/L, corresponding to test item concentrations of 2.2 and 99 mg/L.

 

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

 

Exposure of Daphnia magna to the test item gave the following results based on the nominal loading rates:

Parameter

Endpoint

Concentration (mg/L loading rate WAF)

Immobilisation

EL10

> 100

Immobilisation

EL20

> 100

Immobilisation

EL50

> 100

Immobilisation

No Observed Effect Loading Rate

100

Immobilisation

Lowest Observed Effect Loading Rate

Not determined

Reproduction

EL10

28

(95 % confidence limits 7.4 to 107)

Reproduction

EL20

52

(95 % confidence limits 12 to 223)

Reproduction

EL50

> 100

Reproduction

No Observed Effect Loading Rate

45

Reproduction

Lowest Observed Effect Loading Rate

100

Body length

EL10

71

(95 % confidence limits 33 to 151)

Body length

EL20

> 100

Body length

EL50

> 100

Body length

No Observed Effect Loading Rate

45

Body length

Lowest Observed Effect Loading Rate

100

Supporting study

Guideline: A study was performed to assess the effect of the test material on the reproduction ofDaphnia magnaover a 21 day period. The method followed that described in the OECD Guidelines No 211 "Daphnia magna, Reproduction test".

Methods: Based on the results of an acute toxicity study,Daphnia magnawere exposed (10 replicates of a single daphnid per group) to filtered WAF of the test material over a range of nominal loading rates of 1.5, 4.8, 15, 48, 150 mg/L for a period of 21 days. The test solutions were renewed 3 times per week. The numbers of live and dead adult Daphnia and young daphnids were determined daily. TheDaphniawere fed daily with an algal suspension.

Results: Analysis of the test loading rates at 0 hours showed the measured concentrations to range from 0.106 to 0.826 mg/L whilst analysis of the old or expired media sampled throughout the study showed measured concentrations to range from less than the limit of quantification of the analytical method to 0.863 mg/L. These results were in-line with the recovery and stability analyses performed on the WAFs which showed the test material to be unstable in both light and dark conditions at the lower test concentrations. On the majority of sampling occasions increasing measured concentrations were shown with increasing loading rates.

Given that the toxicity cannot be attributed to any one or more of the components in the WAF, but to the mixture as a whole, the results are based on nominal loading rates only.

The 14 and 21 -day EL50 (immobilisation) values, based on the nominal loading rates, for the parentalDaphniageneration were calculated to be 48 and 27 mg/L loading rate filtered WAF with 95% confidence limits of 33 -69 mg/L loading rate filtered WAF and 15 -48 mg/L loading rate filtered WAF respectively.

The 21 -day EL50 (reproduction) was calculated to be 31 mg/L loading rate filtered WAF with 95% confidence limits of 37 -37 mg/L.

The LOEC is considered to be 48 mg/L loading rate filtered WAF on the basis that at this loading there were significant mortalities (immobilisation) observed in the parental generation.

The NOEC is considered to be 15 mg/L loading rate filtered WAF on the basis that at this loading rate there were no mortalities (immobilisation) observed in the parental generation and that there were no significant differences between the control and the 15 mg/L loading rate filtered WAF test group in terms of the number of live young produced per adult by day 21.