Registration Dossier

Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2003-03-21 to 2003-04-11
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
A water hardness value which falled 1 mg/L below the guideline specification minimum, however this deviation was not considered to be of biological significance.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Tri (hexyl, octyl, decyl) citrate
- Substance type: pure active substance
- Physical state: liquid
- Storage condition of test material: at room temperature
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Melting point: -29°C (freezing point: -35.5°C)
- Boiling point: test substance showed decomposition above 260°C
- Vapour pressure: 8.5 x 10E-3 p at 25°C
- Water solubility (under test conditions): < 1 mg/L
- Solubility in organic solvents: soluble
- log Pow: > 6
- pKa: not applicable
- Base or acid catalysis of test material: no data
- UV absorption: no data
- Stability of test material at room temperature: stable
- pH dependance on stability: no data


OTHER PROPERTIES (if relevant for this endpoint)
- Adsorption characteristics: no data

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
A preliminary stability trial was conducted to assess the stability of Tri (hexyl, octyl, decyl) citrate in acetone Solvent stock solutions were prepared at 0.1, 0.98 and 1 mg/mL by direct addition of the test substance to acetone. Samples of each solvent stock were taken for chemical analysis at 0 and 24 hours. The mean measured concentration of test substance in the preliminary trial were 0.08, 0.90 and 0.81 mg/mL respectively, corresponding to 80, 92 and 81 % of the nominal concentrations.
During the main test:
- Concentrations: Stock solutions with 0.03, 0.1, 0.31, 0.98, 3.13, 10 mg test substance/mL acetone, recovery: 111, 89, 86, 88, 88, 87 % of the nominal concentrations. The concentration of the test substance in test water was not determined as the test article had a negligible solubility in water.
- Sampling method: Samples were taken for the chemical analysis of the test substance from freshly prepared and 24 hours old solvent stock solutions used for the preparation of test media, on three occasions during the study.
- Sample storage conditions before analysis: not mentioned

Test solutions

Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Solvent stocks were prepared by direct adding of the test substance to acetone in individual volumetric flasks. Each of the test media at nominal test substance exposure concentrations were prepared by adding 100 µL of the approprate solvent stock to individual 1L volumetric flask made up to volume with test water. Each volumetric flaks was shaken for approx. 1 minute before dispensing test media to the test vessels.
Fresh stock solutions were prepared on days 0, 3, 5, 7, 10, 12, 14, 17 and 19 of the study.
- Controls: test water (control) and test water containing solvent at 0.1 mL/L (solvent control)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 0.1 mL/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Freshly prepared test media of 0.003, 0.01, 0.031, 0.098 and 0.313 mg/L appeared as clear and colourless solutions. At the highest concentration of 1 mg/L the test solutions appeared as clear and colourless non-homogenuous liquid dispersion with particular matter on the base of the test vessels. Old test media following daily renewal (days 1 to 21) appeared as feint green-brown solutions with seaweed and algal particulate settling on the base of the test vessels at all test substance concentrations.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Strain/clone: Daphnia magna
- Source: Sheffield University, Sheffield, UK, subsequently in-house breeding
- Age of parental stock (mean and range, SD): less than 24 hours, no further details mentioned
- Feeding during test
- Food type: Chlorella vulgaris and seaweed extract of Ascophyllum nodusum (Marinure(R), Glenside Organics Ltd., Stirling, Scotland)
- Amount: 5 x 10E5 cells/mL chlorella vulgaris, 0.2 mL of a seaweed suspension of 16 mL/L in distilled water
- Frequency: daily


ACCLIMATION
- Acclimation period: Juveniles to be used in the toxicity test were collected from at least second brood onwards from same age cultures. The day before the start of the test, juveniles present in the parental culture were removed and discarded. At the start of the test, juveniles (less than 24 hous old) were removed from the parental culture and transferred to a holding vessel in fresh culture medium. The juveniles were left for at least one hour before selecting actively swimming individuals for use.
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: same as test
- Feeding frequency: same as test
- Health during acclimation (any mortality observed): not mentioned

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
none

Test conditions

Hardness:
169 - 170 mg/L CaCO3
Test temperature:
20.2 - 20.8 °C
pH:
7.6 - 7.8
Dissolved oxygen:
98 - 102 % air saturation
Salinity:
192 mg//L NaHCO3; 120 mg/L CaSO4 x 2 H2O; 246 mg/L MgSO4 x 7 H2O; 8 mg/L KCl
Nominal and measured concentrations:
Nominal: 0.003, 0.01, 0.031, 0.098, 0.313, 1 mg/L
Nominal concentrations of stock solution: 0.03, 0.1, 0.31, 0.98, 3.13, 10 mg/L
Mean measured concentrations of stock solutions (after 0 and 24 h): 0.0334, 0.0893, 0.267, 0.863, 2.74, 8.66 mg/mL
As the measured concentrations of the test substance in the solvent stocks were between 80 and 120% of the nominal concentrations, the evaluation was based on nominal concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): not mentioned
- Material, size, headspace, fill volume: 100 mL glass beaker containing 50 mL test medium
- Aeration: no
- Renewal of test solution (frequency/flow rate): daily
- 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): 10
- Biomass loading rate: 1 organism/50 mL


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: standard hard water prepared with AnalaR grade reagents and reverse-osmosis water
- Alkalinity: 77 - 85 mg/L (as CaCO3)
- Ca/mg ratio: not mentioned
- Conductivity: not mentioned
- Salinity: 192 mg//L NaHCO3; 120 mg/L CaSO4 x 2 H2O; 246 mg/L MgSO4 x 7 H2O; 8 mg/L KCl
- Culture medium different from test medium: no
- Intervals of water quality measurement: not mentioned


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hour light
- Light intensity: not mentioned (fluorescent lightning)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable): At 24 hour intervalls, the adult Daphnia were observed for the presence of eggs in the brood sac (gravid or non-gravid) and mortalitiy. Any juvenile Daphnia produced (live of dead) in the previous 24 hour period were counted, recorded and discarded. The pH, temperature and concentration of dissolved oxygen were determined in each freshly prepared test medium at each renewal and also from pooled samples of the old test media from the replicat test vessels at each of the test concentrations. The total hardness and alkalinity of the medium was determined for each batch prepared. The ambient, minimum and maximum temperatures of the dilution medium in a vessel adjacent to the test vesssels was recorded daily. Observations were made on the appearance and behaviour of the test substance in the solvent stock solutions and the test media at the start and end of each exposure period.


VEHICLE CONTROL PERFORMED: yes


RANGE-FINDING STUDY
- Test concentrations:
- Results used to determine the conditions for the definitive study:
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
21 d
Dose descriptor:
LC50
Effect conc.:
0.063 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.003 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.01 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.194 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.098 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.313 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
- Mortality of parent animals: see table below
- Mean cumulative number of live juveniles produced per exposure concentration after 21 days: see table below
- Body length and weight of parent animals: not determined
- Time to first brood release: on day eight
- Other biological observations: first adult Daphnia magna mortality observed on day 3
Reported statistics and error estimates:
The 21 day EC50 and LC50 values were calculated using probit analysis after Log10 transformation of the nomimal exposure concentrations. The cumclative total number of live juveniles per surviving adult for the 21 days of the test was calculated. The juveniles produced by adults that died during the study were not included in the calculations of the mean number of juveniles produced or in the statistical analysis.
A Levene's test for equality of variances across the treatments was performed followed by an Analysis of Variance (Dunnett's test) was performed on the cumulative number of live juveniles produced per treatment at day 21.
No statistically significant difference was seen in the number of live juvelines per survivng adult in the control and solvent control treatments (p¿0.05). The mean cumulative number of live juveniles produced per exposure contentration after 21 days from the surviving adults was in the test substance concentration of 0.313 mg/L significantly different from the control treatments (p>0.05).

Any other information on results incl. tables

Table #1: Cumulative adult Daphnia magna mortality at Day 21

Nominal concentration [mg/L]   % mortality of adult Daphnia magna after 21 days
Control   10
 Solvent control  10
 0.003  10
 0.01  40
 0.031  70
 0.098  10
 0.313  60
 1  100

Table #2: Mean cumulative number of live juveniles produced per exposure concentrations after 21 days from the suviving adult Daphnia magna

Nominal concentration[mg/L]   Mean cumulative number of live juvenilesafter 21 days  Mean cumulative number of dead juvenilesafter 21 days
 Control  116  0.556
 Solvent control  159  0.889
 0.003  143  1.00
 0.01  119  0.167
 0.031  136  1.33
 0.098  125  1.00
 0.313  39.0*  0.250
 1  - **  - **

* significantly different form controls (P<0.05) ** cannot be calculated as no adults survived

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Mean number of juveniles per adult in the control treatments 60, adult mortality in controls < 20%
Executive summary:

Based on adult Daphnia magna mortality, the 21 day LC50 for Daphnia magna exposed to the related substance Tri (hexyl, octyl, decyl) citrate was calculated to be 0.0633 mg/L. Base on the cumulative adult mortality after 21 days, the highest no observed effect concentration (NOEC) of Tri (hexyl, octyl, decyl) citrate was observed to be 0.003 mg/L, and the lowest observed effect concentration (LOEC) was observed to bei 0.01 mg/L.

The 21 day reproduction EC50 for Tri (hexyl, octyl, decyl) citrate to Daphnia magna based on the mean number of live juveniles per surviving adult was calculated to be 0.194 mg/L. Base on the production of live juveniles after 21 days the highest no observed effect concentration (NOEC) was caculated to be 0.098 mg/L and the lowest observed effect concentration (LOEC) was calculated to be 0.313 mg/L.