Registration Dossier

Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 JANUARY 2014 – 08 MARCH 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study was conducted in compliance with agreed protocols, including chemical analysis of test material. However, the analytical methods employed for this complex chemistry were unable to reliably measure the test material in water so results were based on nominal loading rates, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Physical state: amber coloured slightly turbid liquid
- Storage condition of test material: room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes

Test solutions

Vehicle:
no
Details on test solutions:
100 mg of test item was added to the surface of 10 liters of test water to give a 10 mg/L loading rate. The test water was then stirred by magnetic stirrer using stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture 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 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 WAF showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first approx. 75-100 mL was discarded) to give the 10 mg/L loading rate WAF. A series of dilutions was made from this WAF to give further test concentrations of 0.010, 0.032, 0.10, 0.32, 1.0, and 3.2 mg/L loading rate WAF.

Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

The concentration and stability of the test item in the test preparations were verified by chemical analysis on Days 0, 1, 3, 4, 7, 8, 14, 15, 20, and 21.
Water samples were taken from the control and each surviving test group (replicates were pooled) for quantitative analysis. Samples of the fresh test preparations were taken on Days 0, 3, 7, 14, and 20 and of the expired test preparations on Days 1, 4, 8, 15, and 21. Samples were stored frozen prior to analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.

Observations on the test media were carried out during the mixing and testing of the WAF. At the start of each mixing period the 10 mg/L loading rate was observed to be a clear colorless water column with oily globules of test item floating at the water surface. After 23 hours stirring and a 1-hour standing period the 10 mg/L loading rate was observed to remain a clear colorless water column with oily globules of test item floating at the water surface. 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.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
Adult Daphnia were maintained in 150 mL glass beakers

Medium: Elendt M7 medium
Temperature: 20 degree C (controlled)
Lighting cycle: 16 hr light, 8 hr dark with 20 minute dawn and dusk transition periods
Food: Mixture of algal suspension (Desmodesmus subspicatus) and Tetramin flake food

Culture conditions ensured that reproduction was by parthogenesis. 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.

Study design

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

Test conditions

Hardness:
160 to 232 mg/L CaCO3
Test temperature:
20 degree C
pH:
7.9 +/- 0.3
Nominal and measured concentrations:
Nominal: 0.010, 0.032, 0.10, 0.32, 1.0, and 3.2 mg/L loading rate WAF.

Details on test conditions:
At 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 ten replicate test vessels were prepared. The test vessels were not aerated.

The control group was maintained uner identical conditions but not exposed to the test item.

The test preparations were renewed daily throughout the 21-day exposure period. 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 2 to 5 uL of a combination of algal and food suspension every day. Equal amounts of food were given to each daphnid.

Each day the numbers of live and dead Parental (P1) generation, the numbers of Filial (F1) daphnia, and the numbers of discarded unhatched eggs were counted. The general condition and size of the parental daphnia was assessed and compared with that of the controls.

The number of daphnids 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 daphnids that were unable to swim for approximately 15 seconds after gentle agitation (i.e., immobile) were considered to be dead. An immobilization criterion for the young daphnids was considered to be inappropriate due to the large numbers of offspring produced in the flasks. At the end of the test, the length of each surviving parent animal was determined.
 
A prestudy was performed to validate the mixing period. The results from this work indicate that there was no significant increase in the measured test concentrations by extending the preparation period for longer than 24 hours.

The pH was measured using a hach HQ30d Flexi handheld meter and the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

Results and discussion

Effect concentrationsopen allclose all
Duration:
21 d
Dose descriptor:
EL10
Effect conc.:
ca. 4.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
other: EL20
Effect conc.:
ca. 5
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
ca. 7.8
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
LOELR
Effect conc.:
ca. 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
ca. 3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Details on results:
All validation criteria were achieved during the test.

No significant mortalities occurred at any test concentrations throughout the test, therefore, the 21-day EL50 (immob) value based on nominal loading rates was observed to be greater than 10 mg/L loading rate WAF.

There was significant effect on size and color of the daphnids in that all of the surviving daphnids on Day 21 at the loading rate of 10 mg/L were markedly smaller and paler in color than control daphnids. After 21 days the length of each surviving adult was determined and demonstrated that there were statistically significant differences (P>= 0.05) between the control and the 10 mg/L loading rate WAF test group in terms of length of the daphnids after 21 days exposure to the test item.

At loading rate WAF test groups 0.010 to 3.2 mg/L there were no statistically significant differences when compared to the control after 21 days in terms of the number of live young produced per adult. The 10 mg/L test group showed a statistically significant difference from the control after 21 days in terms of producing fewer numbers of live young per adult.

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

The Maximum Acceptable Toxicant Loading Rate (MATL) was calculated to be 5.7 mg/L.

Analysis of freshly prepared 3.2 and 10 mg/L loading rate WAFs on days 0, 3, 7, 14, and 20 showed measured concentrations ranged from 0.0237 to 0.0523 mg/L and 0.0732 to 0.118 mg/L, respectively. Analysis of the expired test solutions for 3.2 and 10 mg/L loading rate WAFs on days 1, 4, 8, 15, and 21 showed measured concentrations ranges from less than the limit of quantitation (LOQ), determined to be 0.016 mg/L, to 0.0252 mg/L and less than the LOQ to 0.0917 mg/L, respectively. The dissolved test item may have been more than one or several components of the test item. Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.
Reported statistics and error estimates:
Statistical analysis:

EL50 (immob) values up to Day 21 of the test were estimated by inspection of the data.

EL50 (repro) values after 21 days were calculated by the Linear Interpolation method using the ToxCalc computer software package.

LOEL and NOEL were estimated by the numbers of live young produced per adult over the duration of the test for the control and each test group were compared using one-way analysis of variance incorporating Bartlett's test for homogeneity of variance and the WIlliams test for differences between treatment means when several dose levels are compared with a zero dose control. Results from the control and each test groups daphnia length data, were compared using one way analysis of variance incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several treatments with a control. All statistical analyses were performed using SAS computer software package.

Any other information on results incl. tables

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

 

 

 

 

LoadingRatemg/L

 

 

%

Survival of P1

Number of Live Young

 

Total

Per Parent Surviving to the End of the Test

Control

90

821

91

0.010

100

982

98

0.032

100

999

100

0.10

90

969

96

0.32

100*

844

94

1.0

100

1046

105

3.2

90

950

105

10

80

200

25

 * One accidental adult mortality, therefore results based on 9 adult daphnia

 

 

LoadingRatemg/L

NumberofDeadYoung

NumberofUnhatchedEggs

 

 

Total

 

PerFemale(cumulative)

 

Total

 

PerFemale(cumulative)

Control

0

0

0

0

0.010

0

0

0

0

0.032

0

0

0

0

0.10

0

0

0

0

0.32

0

0

0

0

1.0

0

0

0

0

  3.2  0  0
  10   0   0   0   0

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Daphina magna were exposed to the test item for 21 days and did not result in any significant mortalities at all the test concentrations employed during the test. The 21 day EL50 (immobilization) value, based on nominal test concentrations for the parental Daphnia generation (P1) was estimated to be greater than 10 mg/L loading rate WAF. The 21 -Day EL50 (reproduction) value, based on nominal test concentrations was 7.8 mg/L loading rate WAF. The "lowest observed effect loading rate" (LOEL) and the "no observed effect loading rate" (NOEL) based on nominal loading rates were 10 and 3.2 mg/L, respectively. The "maximum acceptable toxicant loading rate" (MATL) was calculated to be 5.7 mg/L loading rate WAF.

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 (2004) No 211, "Daphnia magna Reproduction Test" referenced as Method C.20 of Commission Regulation (EC) No. 440/2008 and the US EPA Draft Ecological Effects Test Guidelines OCSPP 850.1300 "Daphnid Chronic Toxicity Test".

 

Methods

 

Based on the results of an acute toxicity test to Daphnia magna, (Harlan Laboratories Ltd, Study Number 0525/0979), Daphnia magna were exposed (10 replicates of a single daphnid per group) to Water Accommodated Fractions (WAFs) of the test item over a range of loading rates of 0.010, 0.032, 0.10, 0.32, 1.0, 3.2 and 10 mg/L for a period of 21 days. The test solutions were renewed daily.

 

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

 

Analysis of the freshly prepared 3.2 and 10 mg/L loading rate WAFs on days 0, 3, 7, 14 and 20 showed measured concentrations ranged from 0.0237 to 0.0523 mg/L and 0.0732 to 0.118 mg/L respectively. Analysis of the expired 3.2 and 10 mg/L loading rate WAFs on days 1, 4, 8, 15 and 21 showed measured concentrations ranged from less than the Limit of Quantification (LOQ), determined to be 0.016 mg/L, to 0.0252 mg/L and less than the LOQ to 0.0917 mg/L respectively.

 

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.

 

Conclusion

Exposure of Daphnia magna to the test item resulted in no significant mortalities at all the test concentrations employed during the test. The 21 day EL50 (immobilization) value, based on nominal test concentrations for the parental Daphnia generation (P1) was estimated to be greater than 10 mg/L loading rate WAF. The 21 -Day EL50 (reproduction) value, based on nominal test concentrations was 7.8 mg/L loading rate WAF. The "lowest observed effect loading rate" (LOEL) and the "no observed effect loading rate" (NOEL) based on nominal loading rates were 10 and 3.2 mg/L, respectively. The "maximum acceptable toxicant loading rate" (MATL) was calculated to be 5.7 mg/L loading rate WAF.