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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:
from 2004-05-28 to 2004-08-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented guideline study
Reason / purpose:
read-across: supporting information
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
No test surrogate or analogue material
Analytical monitoring:
yes
Details on sampling:
Samples were analyzed for each new test and control solution on days 0, 7, 14, and 20 (2 individual replicates) and the corresponding old solutions (3 individual replicates) on days 1, 8, 15 and 21.
Microliter aliquots of hydrocarbons, C10 -C13, n-alkanes, isoalkanes,cyclics, aromatics (2 -25%) and 2-methylnaphthalene internal standard solutions in acetone were spiked directly into 10 mL gas tight syringes containing reconstituted water. The syringe contents were transferred to headspace (ca. 20 mL) sample vials containing 5 g sodium sulfate. The vials were crimp sealed and shaken to solubilize the sodium sulfate prior to being placed on the headspace sampler for analysis.
Vehicle:
no
Details on test solutions:
Individual treatments were prepared by adding the appropriate amount of test substance to 13 l of vehicle/dilution water in glass aspirator bottles (capacity 13.8 l). The test substance was added to the water in the aspirator bottles using stainless stell and glass syringes. The syringes were weighed with and without the test substance to determine the actual loading rate. The mixing vessels were sealed with foil covered rubber stoppers; The mixtures were stirred using a 4.4% (of the static liquid depth) vortex for approximately 24 hours on magnetic stirplates with Teflon coated stirbars at room temperature (22.6°C, S.D. 0.3°C). The stirbars were 1.3 cm in diameter and 7.8 cmlong. The depth of solution was 34 cm and the mixing speed was approximately 200 rpm. As stirring initiated and after stirring, all treatments appeared clear/colourless with test substance observed floating at the surface. The mixtures were allowwed to equilibrate to test temperature in a waterbath at 21.8 °C for approximately 1 hour without stirring before removing the WAFs for testing. New WAFs were prepared daily for the renewals.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Source: Cultured at the test facility. Original culture supplied by Aquatic Biosystems, Inc., Fort Collins, Colorado. Stater culture received on 2002-04-11.

HUSBANDRY and ACCLIMATION
Eight daphnids were kept in 1-liter glass culture beakers with approximately 800 ml of reconstituted water (study vehicle/dilution water). The culture chamber was maintained at 20 +/- 1°C under a 16 hour light 8 hour dark photoperiod (10 - 20 foot/candles, 108 - 215 Lux). Day 0 cultures were started daily (at least 5 days per week) using 8 <24h old neonates from culture beakers between 12 and 18 days old, exhibiting <=20% adult immobillization. Cultures were transferred to fresh reconstituted water on regular intervals to ensure that <=24h old neonates are available for studies and to start new cultures.
Cultures of Daphnia magna were fed Pseudokirchneriella subcapitata (approximately 4.5 x 10e+5 cells/ml) and 4.0 ml of a yeast/salmon starter/wheat grass (YTC) mixture per 800 ml of reconstituted water (vehicle/dilution water) daily (5 days per week at a minimum). YTC and algae were supplied by Aquatic Biosystems, Inc., Fort Collins, Colorado.

EXPOSURE
Number: 60
Sex: Female
Age at initiation of exposure: <24h old, taken from 14-day old parents. The pool of neonates had <=10% daily immobilization on the experimental start day.
Daphniids were fed daily by adding 0.5 ml of a 1.3 x 10e+8 cells/ml suspension of Pseudokirchneriella subcapitata to provide approximatley 5.2 x 10e+5 cells/ml. Test organisms were also fed during renewals with 0.05 ml of a 2.5 g/l Microfeast PZ-20 suspension. Total organis carbon analysis of 125 ml of the vehicle/dilution water with 0.5 ml of algae and 0.05 ml Microfeast PZ-20 suspension contained approximatley 0.1 mg C after substracting the carbon content of the vehicle/dilution water. Since the test chambers contained only one daphnid, this equates to approximately 0.1 mg C per daphnid per day. The algae were supplied by Aquatic biosystems, Inc., Fort Collins, Colorado. The Microfeast PZ-20 was supplied by Salt Creek, Salt Lake City, Utah.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
No post exposure
Test temperature:
Mean test temperature: 20.1°C (S.D. = 0.3), continuously monitored by computer in the test area.
pH:
75-8.1
Dissolved oxygen:
6.8-8.6 mg/l
Nominal and measured concentrations:
Loading rates : 0, 0.12, 0.28, 0.65, 1.4, 3.1 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: no headspace
The test chambers were 125-ml capacity clear glass bottles containing approximately 125 ml of solution (no headspace). The test chambers were sealed with foil lined screww type cap to minimize contamination, evaporation and/or volatilization.
- Aeration: no aeration
- Renewal rate of test solution (frequency/flow rate): Test solutions were renewed daily. A separate set of bottles was used on a rotating basis for renewals. Feed was first added to each bottle. There were then filled with the appropriate WAF and immediately capped. The adult D.magna from the old test solution were transferrred to their respective test chamber using a glass pipette, minimizing the amount of time the chambers were left uncapped. The volume of solution transferred was minimized.
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- Biomass loading rate: 1 daphnid par 125 ml

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (see § Any other information on materials and methods below)
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod:16h light/8 hours dark
- Light intensity: 17 to 18 µE.me-2.se-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Immobilization at 24-h intervals
- Presence, appearance and number of neonates daily
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
EL10
Effect conc.:
0.316 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 95% Lower CI = 0.196 mg/l
Key result
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
1.19 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 95% Lower CI = 0.980 mg/l
Duration:
21 d
Dose descriptor:
EL10
Effect conc.:
0.879 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Remarks:
(adults)
Remarks on result:
other: 95% CI = 0.243-1.24 mg/l
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
1.62 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Remarks:
(adults)
Remarks on result:
other: 95% CI = 1.26-2.51 mg/l
Duration:
21 d
Dose descriptor:
LOELR
Effect conc.:
0.65 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
0.28 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOELR
Effect conc.:
3.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Remarks:
(adults)
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
1.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Remarks:
(adults)
Duration:
21 d
Dose descriptor:
LOELR
Effect conc.:
> 1.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
Adult length
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
1.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
Adult length
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
0.109 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 95% Lower CI = 0.054 mg/l
Key result
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.328 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 95% Lower CI = 0.280 mg/l
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
0.248 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Remarks:
(adults)
Remarks on result:
other: 95% CI = 0.061-0.341 mg/l
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.423 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Remarks:
(adults)
Remarks on result:
other: 95% CI = 0.339-0.732 mg/l)
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.203 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.097 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.833 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Remarks:
(adults)
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.372 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Remarks:
(adults)
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 0.372 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
Adult length
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.372 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
Adult length
Details on results:
See below tables 2-4
Results with reference substance (positive control):
No reference substance
Reported statistics and error estimates:
The reproductive Effect Loading (EL) and Effect Concentration (EC) values were calculated using the Power Model of the Benchmark Dose (BMD) method. The adult immobilization Effect Loading (EL) and Effect Concentration (EC) values were determined using a maximum likelihood analysis based on D. J. Finney,1971. The NOELR and NOEC values were determined using Wilcoxon Rank Sum with Bonferroni Adjustment for the growth data and a t-test with Bonferroni Adjustment to analyze the reproduction data, and Fisher's Exact Test for the adult immobilization data. TOXSTAT software10 was
used to determine the NOELR and NOEC values.

Table2 - Analytical chemistry results

 

Nominal concentration (mg/l)

Replicate

Measured concentrations (mg/l)

Average

Day0

New

Day 1

Old

Day 7

New

Day 8

Old

Day 14

New

Day 15

Old

Day 20

New

Day 21

Old

Control

1

0

0

0

0

0

0

0

0

0

2

0

0

0

0

0

0

0

0

3

0

0

0

0

0

0

0

0

0.12

1

0.0417

0.0337

0.0357

0.0306

0.0451

0.0334

0.0403

0.0358

0.0390

±0.0038

2

0.0456

0.0421

0.0389

0.0377

0.0429

0.0405

0.0398

0.0393

3

n/a

0.0384

n/a

0.0376

n/a

0.0417

n/a

0.0383

0.28

1

0.105

0.0905

0.105

0.0916

0.110

0.0849

0.120

0.0907

0.0969

±0.0133

2

0.113

0.0852

0.102

0.0880

0.109

0.0893

0.121

0.0711

3

n/a

0.0846

n/a

0.0858

n/a

0.0907

n/a

0.0997

0.65

1

0.215

0.186

0.225

0.189

0.236

0.217

0.230

0.175

0.203

±0.021

2

0.205

0.179

0.209

0.191

0.231

0.218

0.203

0.172

3

n/a

0.179

n/a

0.183

n/a

0.222

n/a

0.189

1.4

1

0.407

0.334

0.376

0.362

0.365

0.382

0.380

0.323

0.372

±0.036

2

0.434

0.373

0.417

0.377

0.425

0.358

0.373

0.285

3

n/a

0.333

n/a

0.395

n/a

0.382

n/a

0.354

3.1

1

0.904

0.729

No samples due to complete immobilization

0.833

±0.085

2

0.36

0.791

3

n/a

0.807

 

 

Table 3- Reproduction, survival and length endpoints

21-day effect loading (EL10and EL50) based on reproduction

 

EL10

EL50

 

0.316 mg/l (0.196 mg/l)*

1.19 mg/l (0.980 mg/l)*

 

21-day effect loading (EL10and EL50) based on adult immobilization

 

EL10

EL50

 

0.879 mg/l (0.243-1.24 mg/l)**

1.62 mg/l (1.26-2.51 mg/l)**

 

Reproduction

Adult immobilization

Adult length

LOELR

NOELR

LOELR

NOELR

LOELR

NOELR

0.65 mg/l

0.28 mg/l

3.1 mg/l

1.4 mg/l

>1.4 mg/l

1.4 mg/l

 

21-day effect concentration (EC10and EC50) based on reproduction

 

EC10

EC50

 

0.109 mg/l (0.054 mg/l)*

0.328 mg/l (0.280 mg/l)*

 

21-day effect concentration (EC10and EC50) based on adult immobilization

 

EC10

EC50

 

0.248 mg/l (0.061-0.341 mg/l)**

0.432 mg/l (0.339-0.732 mg/l)**

 

Reproduction

Adult immobilization

Adult length

LOEC

NOEC

LOEC

NOEC

LOEC

NOEC

0.203 mg/l

0.097 mg/l

0.833 mg/l

0.372 mg/l

>0.372 mg/l

0372 mg/l

 

* 95% Lower Confidence Interval (the statistical program used does not calculate an upper C.I.)

** 95% Confidence Interval

 

Table 4- Summary of survival, reproduction and length results

Loading rate (mg/l)

Adult immobilization (%)

Neonates per adult

Adult average length (mm)

0

10

139

4.0

0.12

10

149

4.0

0.28

0

143

4.0

0.65

20

101

3.7

1.4

40

61

3.7

3.1

100

n/a

n/a

 

Validity criteria fulfilled:
yes
Conclusions:
Chronic toxicity results are expressed as the Effect Loading 50 (EL50), and Effect Concentration 50 (EC50); which are the loading rte and concentration which do not exhibit a statistical difference in reproductive output from the control.

The EL50 based on reproduction was 1.2 mg/l. The EC50 based on reproduction was 0.328 mg/l. Reproduction was more sensitive than growth and survival, resulting in a NOELR of 0.28 mg/l and a NOEC of 0.097 mg/l.
Executive summary:

This study was conducted to assess the chronic affects of the water-accomodated fractions (WAFs) of hydrocarbons, C10 -C13, n-alkanes, isoalkanes,cyclics, aromatics (2 -25%) (MRD-04 -991), to the daphnid, Daphnia magna, in a 21-day semi-static (renewal) test conducted according to OECD guideline 211.

Individual treatments were prepared by adding the appropriate amount of test substance to 13 l of vehicle/dilution water in glass aspirator bottles (capacity 13.8 l) and stirring on magnetic stirplates with a vortex of approximately 4% of the static liquid depth for approximately 24 hours. Approximately 1 hour after stirring was hakted, the aqueous portions (water accommodated fractions) of each treatment solution were removed for testing. The actual loading rates were 0 (control), 0.12, 0.28, 0.65, 1.4 and 3.1 mg/l. The mean measured concentrations were ND (Not detected; control), 0.039, 0.097, 0.203, 0.372 and 0.833 mg/l.

Ten replicates per treatment were tested. One <24 -hour old daphnid was added to each replicate. Replicate chambers were 125 ml capacity glass bottles, closed using screw-type tops with foil liners (the chamber contained no headspace). Water quality parameters (temperature, pH, and dissolved oxygen) were within acceptable limits throughout the testing period. Observations for immobilization were performed and recorded at approximately 24 -hour intervals after the begining of the test. The adults were transferrred to fresh solution daily and the numbers recorded. The control daphnids released their first brood on day 8 and 9. The coefficient of variation for control fecundity was 17%. At the end of the test, the total number of living offspring produced per live parent animal was detrmined. Adult organisms were measured (body length excluding the anal spine) at termination in order to determine if growth effects occurred.

Chronic toxicity results are expressed as the Effect Loading 50 (EL50), and Effect Concentration 50 (EC50); which are the loading rte and concentration which do not exhibit a statistical difference in reproductive output from the control.

The EL50 based on reproduction was 1.2 mg/l. The EC50 based on reproduction was 0.328 mg/l. Reproduction was more sensitive than growth and survival, resulting in a NOELR of 0.28 mg/l and a NOEC of 0.097 mg/l.

Description of key information

Key value for chemical safety assessment

Additional information

A study was performed on Daphnia magna exposed to hydrocarbons, C9 -C12, n-alkanes, isoalkanes,cyclics, aromatics (2-25%). The 21-d NOELR based on reproduction was found to be 0.28 mg/l.