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Long-term toxicity to aquatic invertebrates

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long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-11-29 to 2005-12-20
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study
according to guideline
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
Analytical monitoring:
Details on sampling:
- Concentrations: Newly prepared test solutions on test days 0, 7, 14 and 20; aged exposure solutions on test days 1, 8, 15 and test termination (day 21).
Details on test solutions:

- Method: The primary stock solution was prepared by quantitatively transferring the entire amount of test substance received to a 25-mL volumetric flask and bringing it to final volume with acetone. Triplicate 25.0-µL aliquots of the stock solution were then assayed by liquid scintillation counting (LSC). Based on this analysis and the Study Sponsor supplied specific activity of 56.333 mCi/g (125,100 dpm/µg), the stock solution was determined to have a concentration of 186 mg/L.

Secondary stock solutions were prepared from dilutions of the 186 mg/L primary stock solution at test initiation and weekly thereafter during the toxicity test using the following preparation scheme:

Primary Volume of Total Volume Secondary
Stock Stock Sol. Diluted to Stock
Conc. Used with Acetone Conc.
(mg/L) (mL) (mL) (mg/L)

186 1.370 5.0 51
186 0.700 5.0 26
186 0.350 5.0 13
186 0.172 5.0 6.4
186 0.086 5.0 3.2

All resulting stock solutions were observed to be clear and colourless with no visible undissolved test substance present. Stock solutions were stored in a freezer (-90 to -70 ºC) when not in use.

During the 21-day toxicity test, each test concentration was prepared individually at test initiation and at each renewal interval (i.e., every 24 hours) by injecting 6.5 µL of the appropriate secondary stock solution to each replicate test vessel using a 25-µL gas-tight syringe as follows:

Secondary Volume of Total Volume Nominal
Stock Stock Sol. Diluted to Conc.
Conc. Used w/ Dil. Water
(mg/L) (uL) (mL) (ug/L)

51 6.5 65 5.1
26 6.5 65 2.6
13 6.5 65 1.3
6.4 6.5 65 0.64
3.2 6.5 65 0.32

Each test vessel was slowly inverted three times after toxicant injection to promote mixing.

- Controls: A set of dilution water control vessels containing dilution water only was established. A solvent control solution (0.1 mL/L) was prepared by injecting 6.5 µL of acetone to 65 mL of dilution water. The control and solvent control were maintained under the same conditions as the treatment solutions.
Test organisms (species):
Daphnia magna
Details on test organisms:

- Source: Springborn Smithers culture facility

- Age of parental stock (mean and range, SD): Test organisms were <24 hours old at the initiation of the test. Test organisms were not from the first brood of culture organisms and were derived from healthy culture stock.

- Culture conditions: The daphnid culture area received a regulated photoperiod of 16 hours of light and 8 hours of darkness. Light intensity of 95 to 119 footcandles at the surface of the culture solution was provided by fluorescent bulbs. Culture vessels were maintained in a temperature-controlled water bath designed to maintain the culture solution temperature at 20 ± 2 ºC.

- Culture medium: The culture water was prepared by fortifying well water based on the formula for hard water (U.S. EPA, 1975) and filtering it through Amberlite XAD-7 resin column to remove any potential organic contaminants. This water had total hardness and total alkalinity ranges as calcium carbonate (CaCO3) of 180 to 190 mg/L and 120 to 130 mg/L, respectively, a pH range of 7.8 to 8.3, a temperature range of 18 to 22 ºC, a dissolved oxygen concentration range of 7.0 to 9.8 mg/L, and a specific conductance range of 500 to 600 micromhos per centimetre (µmhos/cm).

- Feeding: In culture, daphnids were fed various amounts based on their age. Daphnids that were 0 to 6 days old were fed 0.5 mL of algae and 0.5 mL of YCT suspension per vessel per day. Daphnids that were 7 to 10 days old were fed 1.0 mL of algae and 0.5 mL of YCT suspension per vessel per day. Daphnids that were > 10 days old were fed 1.5 mL of algae and 0.5 mL of YCT suspension per vessel per day.

- Feeding during test: Daphnids were fed unicellular green algae, Ankistrodesmus falcatus (4 x 107 cells/mL) in addition to a suspension of YCT (yeast, cereal leaves and flaked fish food).
Test type:
Water media type:
Limit test:
Total exposure duration:
21 d
The source water prepared during the definitive exposure was characterised as having a total hardness and a total alkalinity (as CaCO3) of 180 mg/L and 120 mg/L
Test temperature:
18 to 24 ºC
The pH measured in the dilution water control ranged from 8.1 to 8.3 in newly prepared test solutions and from 7.7 to 8.4 in aged solutions during the exposure period.
Dissolved oxygen:
The dissolved oxygen concentration ranged from 7.7 to 10.0 mg/L in newly prepared test solutions and from 6.6 to 11.0 mg/L in aged solutions during the exposure period
Not applicable
Nominal and measured concentrations:
Nominal test concentrations were 0.32, 0.64, 1.3, 2.6 and 5.1 µg/L.

Arithmetic mean measured concentrations: 0.27, 0.57, 1.2, 2.5 and 4.6 µg/L
The results are interpreted with reference to mean measured concentrations
Details on test conditions:

- Test vessel: The toxicity test was conducted in 65-mL screw top GC vials, each filled completely (no headspace) with test solution and sealed with a screw top to minimize the loss of test substance from the test solution.

- Aeration: none

- Renewal rate of test solution (frequency/flow rate):

- No. of organisms per vessel: Daphnids were impartially added to an intermediate beaker by adding no more than one daphnid to each beaker until all beakers contained one daphnid. This procedure was repeated until each intermediate beaker contained eleven daphnids. The test was initiated when each intermediate beaker of eleven daphnids was added (one daphnid per replicate vessel) to an impartially selected treatment level or control.

- No. of vessels per concentration and control (replicates): Eleven replicate test vessels were established for each nominal concentration and the controls (i.e., replicates A through J for the biological measurements and replicate K for analysis of newly prepared test solutions).

- Biomass loading rate: 1 daphnid/65 mL of test medium


- Source/preparation of dilution water: Dilution water chemistry (hardness, alkalinity, pH, TOC): The source water prepared during the definitive exposure was characterised as having a total hardness and a total alkalinity (as CaCO3) of 180 mg/L and 120 mg/L, respectively, a pH range of 7.9 to 8.1, a dissolved oxygen concentration range of 8.0 to 8.7 mg/L and a specific conductance range of 500 to 600 micromhos per centimetre (µmhos/cm). Water quality parameters were measured on each batch of fortified water prior to use. The total organic carbon (TOC) of the dilution water source for November and December 2005 was 0.78 and 0.17 mg/L, respectively.


- Adjustment of pH: no

- Lighting (quality, intensity, and periodicity): Light at an intensity range of 12 to 14 µE·m-2·s-1 at the surface of the exposure solutions was provided by fluorescent bulbs. The test area received a regulated photoperiod of 16 hours of light and 8 hours of darkness. Sudden transitions from light to dark and vice versa were avoided. Light intensity was measured once during the test.

EFFECT PARAMETERS MEASURED: Survival, reproduction and growth (total body length and dry body weight)

Reference substance (positive control):
21 d
Dose descriptor:
Effect conc.:
>= 4.6 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: survival, reproduction and growth
Details on results:
- Mortality of parent animals: 0-10% in Controls

- No. of offspring produced per day per female: 145-168 offspring/parent in Controls

- Time to first brood release or time to hatch: Day 8
Reported statistics and error estimates:
Student's t-Test, Fisher's Exact Test, Chi-Square Test for normality, Bartlett's Test and Williams' Test. No statistically significant effects on survival, growth and reproduction were observed in the test.

Biological observations:

- Number of immobilised daphnids as compared to the number exposed:  Number of immobilised daphnids:  2, Number exposed:  70 (includes control and solvent control).

- Concentration response with 95% confidence limits:  > 4.6 µg/L (empirically estimated) for survival and reproduction, corresponding 95% confidence intervals could not be calculated.


- Survival: 90 and 100% survival was observed in the control and solvent control, respectively (pooled control = 95%).  Survival of 100, 90, 100, 100 and 100% was observed among daphnids exposed to the 0.27, 0.57, 1.2, 2.5 and 4.6 µg/L treatment levels, respectively.  

- Reproduction: Organisms exposed to the control and solvent control released a mean cumulative offspring per female of 165 and 148, respectively (pooled control = 156 offspring per female).  Organisms exposed to the 0.27, 0.57, 1.2, 2.5 and 4.6 µg/L treatment levels released a mean cumulative offspring per female of 158, 151, 141, 149 and 155, respectively.  

- Growth: Total body length at test termination among control and solvent control daphnids averaged 4.91 and 4.84 mm per daphnid, respectively (pooled control = 4.88 mm).  Total body length among daphnids exposed to the 0.27, 0.57, 1.2, 2.5 and 4.6 µg/L treatment levels averaged 4.86, 4.85, 4.84, 4.84 and 4.91 mm, respectively.  

Dry weight at test termination among control and solvent control daphnids averaged 1.06 and 1.12 mg per daphnid, respectively (pooled control = 1.09 mg).  Dry weight among daphnids exposed to the 0.27, 0.57, 1.2, 2.5 and 4.6 µg/L treatment levels averaged 1.10, 1.15, 1.11, 1.17 and 1.18 mg per daphnid, respectively.  

Validity criteria fulfilled:
A 21-day NOEC of ≥4.6 µg/L has been determined for the effects of the test substance on survival, growth and reproduction of Daphnia magna. Since no concentration tested resulted in >50% immobilisation or reduction in reproductive output, the EC50 for both survival and reproduction was empirically estimated to be >4.6 µg/L, the highest mean measured concentration tested and the functional limit of water solubility under the conditions of administration.

Description of key information

Daphnia reproduction: (21 day): NOEC: ≥4.6 µg/l, effects of the test substance on survival, growth and reproduction of Daphnia magna.  

Key value for chemical safety assessment

Additional information

Reliable test results are available for long-term toxicity to aquatic invertebrates.

A 21-day NOEC of  ≥4.6 µg/l has been determined for the effects of the test substance on survival, growth and reproduction of Daphnia magna in accordance with OECD TG 211 and in compliance with GLP (Springborn Smithers, 2009). The results have been obtained under semi-static conditions and are expressed relative to geometric mean measured concentrations of the substance.

In view of the hydrolysis half-life of the test substance and the semi static exposure regime, it is likely that the test organisms were exposed primarily to the tested substance rather than its degradation products.