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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:
2010-05-20 to 2010-08-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Sampling method: Water samples were collected from alternating replicate test chambers of each treatment and control group one and six days prior to test initiation to confirm the operation of the diluter. Water samples also were collected from alternating replicate test chambers at the beginning of the test, at approximately weekly intervals during the test and at the end of the test to determine concentrations of the test substance in the test system. Additionally, stock and water samples were collected from test chambers two days after test termination due to questionable results at test termination. All samples were collected mid-depth and placed directly into 100 ml volumetric flasks and processed immediately for analysis.

- Sample storage conditions before analysis: The samples were processed immediately for analysis.
Vehicle:
yes
Details on test solutions:
Stock solutions were prepared two times during the test. For each preparation, a primary stock solution was prepared in DMF at a nominal concentration of 10000 ng/ml. The primary stock solution was mixed by inversion and appeared clear and colourless. A second primary stock solution was prepared by proportional dilutions at a nominal concentration of 700 ng/ml. Proportional dilutions of the 700 ng/ml primary stock solution were then used to prepare the four additional secondary stock solutions at nominal concentrations of 44, 88, 180 and 350 ng/ml. The secondary stock solutions w ere inverted to mix, and appeared clear and colourless.

The five stock solutions were delivered to the diluter mixing chambers (at a rate of 15.5 μl/minute) where they were mixed with dilution water (at a rate of 155 ml/minute) to achieve the desired test concentrations of 4.4, 8.8, 18, 35 and 70 ng/l. The solvent control was prepared by injecting DMF into the mixing chamber assigned to the solvent control. The concentration of DMF in the solvent control and all treatment groups was 0.1 ml/l.

The test solutions in the test chambers for all treatments and control groups appeared clear and colourless at test initiation and termination.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Source: Daphnid neonates used in the test were less than 24 hours old and were obtained from cultures maintained by the test laboratory.

- Culture water: Adult daphnids were cultured in water from the same source and at approximately the same temperature as used during the test.

- Feeding: During culture and testing, daphnids were fed a mixture of yeast, cereal grass media, and trout chow (YCT), as well as a suspension of the freshwater green alga, Pseudokirchneriella subcapitata. Daphnids were fed three times per day through Day 6 of the test and then were fed four times per day until Day 20 and then once on the last day of the test. At each feeding, each test chamber was fed 0.75 mL of YCT and 1.5 mL of algae. While this amount of feed exceeds the OECD guideline( 2) recommended amount of 0.1 to 0.2 mg C/daphnid/day an excess amount was fed in order to maintain sufficient feed in the flow-through system to support acceptable reproduction rates.

- Test organisms: The four adult daphnids used to supply neonates for the test were held for at least 27 days prior to collection of the juveniles for testing and had produced at least one previous brood. Adult daphnids in the culture had produced an average of at least three young per adult per day over the 7 day period prior to the test. The adults showed no signs of disease or stress and no ephippia were produced during the holding period.

- Allocation of test organisms to treatments: At test initiation the juvenile daphnids were collected from the cultures and indiscriminately transferred one or two at a time to transfer chambers (e g , plastic cups) until each chamber contained five daphnids. Each group of daphnids was then transferred to an indiscriminately assigned test compartment. All transfers were made below the water surface using wide-bore pipettes.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
132-140 mg/l as CaCO3
Test temperature:
20.0-20.2°C
pH:
8.0-8.1
Dissolved oxygen:
≥5.5 mg/l
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 0 (Control), 0 (Solvent control), 4.4, 8.8, 18, 35 and 70 ng/l.

Mean measured test concentrations in treated vessels: 3.6, 7.0, 16, 31 and 47 ng/l.

The results are interpreted with reference to mean measured concentrations.
Details on test conditions:
- Test apparatus: A continuous-flow diluter was used to deliver each concentration of the test substance, a solvent (dimethylformamide) control, and a negative (dilution water) control. Syringe pumps were used to deliver the five test substance stock solutions and dimethylformamide (DMF) for the solvent control Into mixing chambers indiscriminately assigned to each treatment and the solvent control. The pumps w ere calibrated prior to the test. The stock solutions were diluted with well water !n the mixing chambers in order to obtain the desired test concentrations. The flow of dilution water to the mixing chambers was controlled by rotameters, which were calibrated prior to test initiation and at weekly intervals thereafter. The flow of test water from each mixing chamber was split and allowed to flow into two replicate test chambers. The proportion of the test water that was split into each replicate was checked prior to the test and at weekly intervals thereafter to ensure that flow rates varied by no more than +/-10% of the mean for the two replicates The diluter flow rate was adjusted to provide approximately five volume additions of test water ln each test chamber per day. The general operation of the diluter was checked visually at least two times per day during the test and at least once at the beginning and end of the test.

- Test chambers: Test chambers were 25-litre Teflon-lined stainless steel aquaria filled with approximately 22 litre of test solution. The daphnids were held in two test compartments suspended in each of two test chambers. Test compartments were 300 mL glass beakers, approximately 6 5 cm in diameter and l2 cm in height. Nylon mesh screens covered two holes on opposite sides of each test compartment to permit test solution to flow in and out of the compartment. The depth of the test water in a representative test compartment was approximately 8 cm, while the depth of water in a representative test chamber was approximately 29 cm. The test chambers were placed in a temperature-controlled water bath to maintain the target temperature throughout the test period.

- Lighting: Fluorescent light bulbs that emit wavelengths similar to natural sunlight (Colortone®50) were used for illumination of the cultures and test chambers. A photoperiod of l6 hours of light and 8 hours of darkness was controlled with an automatic timer. A 30 minute transition period of low
light intensity w as provided when lights went on and off to avoid sudden changes in lighting. Light intensity was measured at test initiation using a SPER Scientific Model 840006C light meter and was 236 lux over one representative test chamber.

- Temperature: The target test temperature was 20+/-1°C. Temperature was measured in each test chamber at test initiation and termination, and at weekly intervals during the test, using a liquid-in-glass thermometer. Temperature also was measured continuously in one negative control test chamber using a Fulscope ER/C Recorder, which was verified prior to test initiation and weekly during the test using a liquid-in-hand glass thermometer.

- Dissolved oxygen and pH: Dissolved oxygen was measured in alternating replicate test chambers of each treatment and control group at test initiation and termination, and approximately three times per week during the test. Measurements of pH were made in alternating replicate test chambers of each treatment and control group at test inanition and at weekly intervals thereafter. Dissolved oxygen was measured using a Thermo Orion 850A plus dissolved oxygen meter, and measurements of pH were made using a Thermo Orion 525A plus pH meter.

- Hardness, alkalinity and specific conductance: Hardness, alkalinity and specific conductance were measured in alternate replicates of the negative (dilution water) control and the highest test concentration test at test initiation and at weekly intervals thereafter.

- Total organic carbon (TOC): TOC was measured in the dilution water at test initiation and termination.

- Biological Observations and Measurements: Observations of each first-generation daphnid were made daily during the test. At these times, the numbers of dead and immobile daphnids were recorded along with any clinical signs of toxicity (e.g. inability to maintain position in the water column, uncoordinated swimming or cessation of feeding). immobility was defined as a lack of movement, except for minor spontaneous random movement of the appendages. The presence of eggs in the brood pouch, aborted eggs, males or ephippia also were recorded daily. With the onset of reproduction, neonates produced by the first generation daphnids were counted and then discarded every Monday, Wednesday and Friday during the test. The body length and the dry weight of each surviving first-generation daphnid were measured at the end of the test.
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 47 ng/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
and survival
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 47 ng/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
and survival
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 47 ng/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: adult mortality and reproduction
Reported statistics and error estimates:
Analysis of variance (ANOVA) was used to determine whether or not statistically significant differences existed among the experimental groups (p = 0.05). Those treatments that were significantly different from the pooled or solvent control treatments were identified using Dunnett's t-test (p≤0.05). All statistical tests were performed using SAS (6) software.

There were <50% reductions in survival and reproduction at the end of the test and this precluded the calculation of EC50 values for these endpoints.

Table 1. Results of analysis of test media

 

Nominal concentration (ng/l)

Mean measured concentration (ng/L)

Mean measured concentration as percentage of nominal

0 (Control)

<LOQ

<LOQ

0 (Solvent control)

<LOQ

<LOQ

4.4

3.6

82

8.8

7.0

80

18

16

89

35

31

89

70

47

67

 

Table 2. Summary of test results

 

Mean measured concentration (ng/L)

Percentage survival at end of test

Mean number

of young produced per reproductive day

Mean length (mm)

Mean dry weight (mg)

0 (Control)

90

9.2+/-0.53

5.1+/-0.058

1.09+/-0.14

0 (Solvent control)

95

9.9+/-1.49

5.2+/-0.058

1.12+/-0.066

3.6

95

11.5+/-0.30

5.1+/-0.000

1.04+/-0.058

7.0

90

10.5+/-0.34

5.1+/-0.050

1.05+/-0.052

16

100

11.7+/-1.42

5.1+/-0.058

1.00+/-0.058

31

100

11.0+/-1.42

5.1+/-0.058

0.96+/-0.039*

47

85

10.4+/-1.89

5.2+/-0.010

1.02+/-0.081

*Statistically significant difference (p<0.05) compared with pooled control by Dunnett’s test

Validity criteria fulfilled:
yes
Conclusions:
A 21-day EC50 of >47 ng/l and NOEC of ≥47 ng/l have been determined for the effects of the test substance on adult mortality, reproduction and growth of Daphnia magna. The results have been obtained under flow-through conditions and are expressed relative to mean measured concentrations of the substance.

Description of key information

(21 day) NOEC: ≥47 ng/l adult mortality, reproduction and growth Daphnia magna, reliability 1.

Key value for chemical safety assessment

Additional information

A 21 day EC50 value of >47 ng/l and NOEC of ≥47 ng/l have been determined for the effects of the test substance on adult mortality, reproduction and growth of Daphnia magna. The results have been obtained under flow-through conditions and are expressed relative to mean measured concentrations of the substance.