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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental starting date: 29 September 2014; Experimental completion date: 21 January 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)
Qualifier:
according to guideline
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control and each surviving test group (replicates pooled). Samples of the fresh test preparations were taken on Days 0, 6, 9, 13, 16 and 20 and of the expired test preparations on Days 1, 7, 10, 14, 17 and 21.
- Sampling method: Not discussed.
- Sample storage conditions before analysis: Day 1, 9 and 21 samples were analysed on the day of receipt whilst all other were stored frozen prior to analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Nominal amounts of test item (3.2, 10.1, 32, 101 and 320 µL, equivalent to 2.3, 7.4, 23, 74 and 230 mg respectively given that I 00 µL of test was determined to weigh 73 mg) were each
separately added to the surface of 2.3 litres of test water to give the 1.0, 3.2, 10, 32 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. During the stirring phase the vessels were sealed with a minimal headspace. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAFs.
On days 6 and 7 the stirring duration for the WAF preparations was 24 and 22 hours respectively.
The concentration and stability of the test item in the test preparations were verified by chemical analysis on Days 0, 1,6, 7,9, 10, 13, 14, 16, 17, 20 and 21.

- Controls: The control group was maintained under identical conditions but not exposed to the test item.

- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia
- Strain/clone: Not reported
- Justification for species other than prescribed by test guideline: n/a
- Source: In-house laboratory cultures.
- Age of parental stock (mean and range, SD): 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.
- Feeding during test
Each daphnid received approximately 5 to 10 µL of an algal suspension (Desmodesmus subspicatus) and approximately 10 to 20 µL of Tetramin® flake food suspension daily. 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.

ACCLIMATION
- Acclimation period: None reported - The reconstituted water (Elendt M7 medium) used for the definitive test was the same as that used to maintain the stock animals.
- Acclimation conditions (same as test or not): Yes
- Type and amount of food: Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis.
- Feeding frequency: Daily
- Health during acclimation (any mortality observed): None reported

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
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.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
212 - 274 mg/L as CaCO3
Test temperature:
20 - 23 °C
pH:
7.5 - 8.5
Dissolved oxygen:
6.9 - 9.5 mg/O2/l
Salinity:
n/a
Conductivity:
not reported
Nominal and measured concentrations:
Nominal (WAF loading rate): 1.0, 3.2, 10, 32 and 100 mg/L
Measured: Chemical analysis of the test preparations showed measured test concentrations to range from less than the limit of detection (LOD) of the analytical method employed which was determined to be 0.0024 mg/L to 0.0363 mg/L. The majority of the measured concentrations determined were between the LOD and the limit of quantification (LOQ) of the analytical method employed which was determined to be 0.021 mg/L.
Given that 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. Additionally, at the 100 mg/L loading rate, the average measured concentration of test item across the test duration was less than the LOQ (0.02 mg/L).
Details on test conditions:
TEST SYSTEM
- Test vessel: Conical flask
- Type (delete if not applicable): closed - sealed with a ground glass stopper
- Material, size, headspace, fill volume: Glass, 100 ml, no headspace, completely filled
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): n/a
- Renewal rate of test solution (frequency/flow rate): Daily renewal; 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.
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): n/a
- Biomass loading rate: Not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water - Elendt M7 medium
- Culture medium different from test medium: No
- Intervals of water quality measurement: 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 Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer. Measurements were made on one replicate for each test concentration. The temperature was also measured every hour in one replicate of the control using a Testo temperature logger. The water hardness of the control and the highest surviving test concentration in the fresh and old media was measured once per week.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods
- Light intensity: 485 to 778 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): 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.

The number of daphnia with eggs or young in the brood pouch was determined daily. 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 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 test, the length of each surviving parent animal was determined.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using less concentrations than requested by guideline: n/a
- Range finding study: None reported
- Test concentrations: n/a
- Results used to determine the conditions for the definitive study: Based on data supplied by the Sponsor: The WAF preparation period was selected based on the results of the water solubility determinations for the test item and related substances which indicated that a 24-Hour period was sufficient to attain saturation of the test medium.
Reference substance (positive control):
not specified
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:
other: immobilisation and reproduction
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:
other: Immobilisation and reproduction
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
Remarks:
parental Daphnia generation
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
Details on results:
- Behavioural abnormalities: None reported
- Observations on body length and weight: No effects reported
- Other biological observations: No effects reported
- Mortality of control: No significant mortality reported
- Other adverse effects control: None reported
- Abnormal responses: None reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: n/a
- Effect concentrations exceeding solubility of substance in test medium: n/a
Reported statistics and error estimates:
An estimate of the 21-Day EL50 value was given by inspection of the immobilisation data. The EL50 (reproduction) value after 21 days was estimated by inspection of the data.
The EL50 (body length) value after 21 days was estimated by inspection of the data.
For the estimation of the "Lowest Observed Effect Loading Rate" (LOEL) and the "No Observed Effect Loading Rate " (NOEL) the numbers of live young produced per adult over the duration of the test for the control and each test group were compared using Williams Multiple Sequential t-test incorporating Shapiro-Wilk's test on normal distribution and Levene's test on variance homogeneity. Results from the control and each test group's daphnia length data, determined for the surviving daphnids on termination of the test, were also compared using Williams Multiple Sequential t-test incorporating Shapiro-Wilk's test on normal distribution and Levene's test on variance homogeneity. All statistical analyses were performed using the ToxRatPro software package (TOXRAT).
ELx (immobilisation) values based on nominal loading rates were calculated by the Maximum-Likelihood Probit method at 21 days.

The application of the test item to Daphnia magna was considered to have no significant effect on the parental survival, parental body length at the end of the test or reproduction.

There were no significant effects resulting from the exposure of Daphnia magna to the test item in terms of parental mortalities (immobilisation), body length at the end of the test and number of neonates produced by Day21, therefore the Lowest Observed Effect Loading Rate was not determined. Consequently it was not possible to determine the No Observed Effect Loading Rate, this was therefore regarded to be equal to or greater than 100 mg/L loading rate WAF.

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 the "filial" daphnids produced by all the test groups were in the same general condition as the young produced by the controls over the duration of the test.

Young were first produced in the control test group on Day 8 of the test.

Numbers of unhatched eggs and dead young were low in all control and treatment groups surviving to maturation.

Table: Summary of Findings Following the Exposure of Daphnia magna for 21 Days

 Nominal Loading Rate  (mg/L)  Percentage Survival of Parental (P1) Generation  Total Number of Live Young  Number of Live Young Per Parent Surviving to the End of the Test Number of Live Young Per Parent at the Start of the Test Excluding Parental Accidental and/or Inadvertent Mortalities 
 Control  80  989  123.6  123.6
 1.0  70  1064  125.0  125.0
 3.2  100*  1147  127.4  127.4
 10  90  1191  119.7  119.7
 32  90  1181  131.2  131.2
 100  80  995  123.1  123.1

* One daphnia was dropped during transfer to fresh test media on Day 3 of the test. This replicate was considered to be an accidental mortality and was therefore excluded from subsequent analysis.

Validity criteria fulfilled:
yes
Conclusions:
The chronic toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave 21-day EL10 and EL50 values of greater than 100 mg/L loading rate WAF, for effects on mobility and reproduction. The No Observed Effect Loading rate was ≥100 mg/L loading rate WAF.
This study showed that there were no toxic effects at the maximum concentration tested in the definitive test.
Executive summary:

Introduction

A 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, the US EPA Draft Ecological Effects Test Guidelines OPPTS 850.1300 "Daphnid Chronic Toxicity Test" and JMAFF Test Guidelines 2-7- 2-2, Daphnia Reproduction, 2001.

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 a Water Accommodated Fraction (WAF).

Based on data supplied by the Sponsor, Daphnia magna were exposed (10 replicates of a single daphnid per group) to a Water Accommodated Fraction (WAF) of the test item over a range of test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L for a period of 21 days. The test solutions were renewed daily 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 a mixture of algal suspension and Tetramin® flake food suspension.

Results

Chemical analysis of the test preparations showed measured test concentrations to range from  less than the limit of detection (LOD) of the analytical method employed which was determined to be 0.0024 mg/L to 0.0363 mg/L. The majority of the measured concentrations determined  were between the LOD and the limit of quantification (LOQ) of the analytical method employed which was determined to be 0.021 mg/L. Measured concentrations between the LOD and LOQ should be interpreted with caution as the recovery data at this level supports the conclusion that whilst test item can be detected, this is only indicative of test item in solution and is not accurately quantifiable. Given that 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. Additionally, at the 100 mg/L loading rate, the average measured concentration of test item across the test duration was less than the LOQ (0.02 mg/L).

There were no significant effects resulting from the exposure of Daphnia magna to the test item in terms of parental mortalities (immobilisation), body length at the end of the test and number of neonates produced by Day 21, therefore the Lowest Observed Effect Loading Rate was not determined. Consequently it was not possible to determine the No Observed Effect Loading Rate (NOELR), this was therefore regarded to be equal to or greater than 100 mg/L loading rate WAF.

Conclusion

The application of the test item to Daphnia magna was considered to have no significant effect on the parental survival, parental body length at the end of the test or reproduction.

The 21-Day EL50 (immobilisation) value, based on nominal loading rates, for the parental Daphnia generation (P1) was estimated to be greater than 100 mg/L loading rate WAF.

The 21-Day EL50 (reproduction) value, based on nominal loading rates, was estimated to be greater than 100 mg/L loading rate WAF.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

Description of key information

No long-term aquatic invertebrate data exist for the registered substance. Reliable data do exist for a related Fischer-Tropsch process-derived product in the relevant carbon number range.

The chronic toxicity of Hydrocarbons, C9-C12 n-alkanes, isoalkanes, <2% aromatics (GS170), to the freshwater invertebrate Daphnia magna has been investigated and gave 21-day EL10 and EL50 values of greater than 100 mg/L loading rate WAF, for effects on mobility and reproduction.  The No Observed Effect Loading rate was ≥100 mg/L loading rate WAF.

This study showed that there were no toxic effects at the maximum concentration tested in the definitive test. This result is read across to the registered substance Hydrocarbons, C9-C11, n-alkanes, isoalkanes, <2% aromatics.

Key value for chemical safety assessment

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