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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:
28 July to 18 Aug 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conditions were not stably maintained, some analytical measurements were questionable, and substantial amounts of test substance were lost between renewals.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report date:
1993

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 202, as promulgated for 1984 (superceded by OECD TG211)
Deviations:
yes
Remarks:
Concentration of test substance was not stably maintained. Oxygen saturation and pH differed from test norms on occasion. Measured concentration of test substance on day 8 was questionable.
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Isooctyl acrylate
EC Number:
249-707-8
EC Name:
Isooctyl acrylate
Cas Number:
29590-42-9
Molecular formula:
C11H20O2
IUPAC Name:
6-methylheptyl acrylate
Details on test material:
- Name of test material (as cited in study report): Isooctyl acrylate (CAS No. 29590-42-9)
- Substance type: Pure substance
- Physical state: Liquid
- Analytical purity: 99.75%
- Lot/batch no.: Lot 3290
- Stability under test conditions: Stable in water
- Storage condition of test material: Stored at room temperature in an amber glass bottle and sealed in a metal container

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Sampling method: Samples (100 mL) were taken daily of the stock solution using volumetric flasks and a preconditioned glass syphon. Samples were analyzed in duplicate. Test solutions (before renewal) were sampled before renewal (no feed had been added) on days 2, 8, 15, and 20. Individual samples were taken of each test vessel on days 3, 9, 16, and 21, immediately following biological observations and water chemsitry determinations (samples contained feed). Pre- and post-renewal samples were taken by mass using a top-loading balance and placed in 125-mL brown glass bottles. After extraction and elution in methylene chloride, extracts were concentrated to 1 mL under a nitrogen stream before GC-MS analysis.

- Sample storage conditions before analysis: Samples analyzed daily except stock solution for days 10-12. These were stored frozen until analysis day 13.

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test substance (110 µL) was added to 8-L well water and stirred vigourously for 20 minutes at ambient temperature in a preconditioned 19-L carboy. After stirring, the mixture was allowed to settle for 20 minutes. The first 100 ml of aqueous solution was removed by syphon and discarded. The remaining solution was considered to have a nominal concentration of 10 mg/L. The pH of the test substance at the nominal high test concentration (8.0 mg/L) was 8.2 and was not adjusted.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None used

- Evidence of undissolved material (e.g. precipitate, surface film, etc): None noted, although it was suggested that an invalidated stock solution sample contained undissolved test substance.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Strain: Not reported
- Source: Stock culture maintained at testing lab
- Age at study initiation: < 24 hours
- Feeding during test: Daily at renewal. Each chamber recieved 3 mL of a mixture of yeast, Cerophyl®, and fermented trout chow (1,800 mg/L total solids), and 6 ml of Pseudokirchneriella subcapitata suspension (3.5E+07 cells/mL)

ACCLIMATION
- Acclimation period: Gravid daphnids were isolated in dilution water containing food 24 h before test initiation. Neonates were obtained at the end of this period
- Health during acclimation (any mortality observed): The stock culture organisms from which the gravid daphnids were obtained and the neonate test organisms appeared to be free from any disease or stress.

Study design

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

Test conditions

Hardness:
128 - 170 mg/L (as CaCO3)
Test temperature:
20.2 - 22.4 °C
pH:
7.6 - 8.6
Dissolved oxygen:
4.5 - 9.0 mg/L
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 0.0, 0.25, 0.5, 1, 2, and 4 mg/L
Mean measured concentrations used for 21-day reproduction EC50: <0.04, 0.13, 0.29, 0.51, 1.06, 2.40 mg/L.
Mean measured concentrations are based on pre-renewal samples taken days 2, 15, and 20 and post-renewal samples taken days 3, 16, and 21

SEE TABLE 2
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL borosilicate glass jar
- Type : closed (sealed with clear plastic wrapping)
- Material, size, headspace, fill volume: 500 mL borosilicate glass jar containing 400 mL medium. Headspace ca. 100 mL.
- Aeration: 100 mL headspace purged with >99.5% purity medical grade oxygen sufficient to inflate plastic seal.
- No. of organisms per vessel: Ten parental organisms
- No. of vessels per concentration (replicates): Four
- No. of vessels per control (replicates): Four
- Other: In addition, one monitoring chamber was assembled for measurement of pH, DO, specific conductance, and temperature (0.0, 0.25, 1, and 4 mg/L concentrations only)

- Source/preparation of dilution water: Well water from the Two Harbors, (Minnesota) area.
- Total organic carbon: Not reported, but COD was 9 mg/L
- Particulate matter: < 4 mg/L
- Metals:
Calcium, 46.5 mg/L
Copper, 13 µg/L
Iron, 3 µg/L
Magnesium, 16.3 mg/L
Sodium, 6.5 mg/L
Zinc, 30 µg/L

- Pesticides: none above detection limit.
- Chlorine: Not reported
- Alkalinity: 105 mg/L to 151 mg/L (as CaCO3)
- Ca/mg ratio: 2.85
- Conductivity: between 280 µmhos/cm and 381 µmhos/cm

- Intervals of water quality measurement: Annually (most recent was August-September 1991)

OTHER TEST CONDITIONS
- Adjustment of pH: no

- Photoperiod: 16h:8h light:darkness. An opaque sheet was suspended approximately 25 cm above the top of the test chambers to decrease direct light intensity.

- Light intensity: The light intensity determined at a later data under conditions identical to the test conditions was less than 10 ft-c (108 lux).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobilization: assessed daily, immobilized parental organisms were removed at medium renewal
Live offspring: counted daily, discarded at solution renewal
Dead offspring/non-emergent eggs: counted daily after live neonates were removed


RANGE-FINDING STUDY
- None
Reference substance (positive control):
no

Results and discussion

Effect concentrations
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.065 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: NOEC for reproduction was calculated as one-half of the LOEC. 21-day NOEC (immobilization), 1.06 mg/L.
Details on results:
DETAILS ON RESULTS:
- No. of live offspring produced per day: See Table 3
- No. of dead offspring produced per day: See Table 4
- Time to first brood release or time to hatch: First neonates appear on day 7 in controls and 0.13, 0.29 and 0.51 mg/L test concentrations
- Immobilization of parent animals: See Table 5

- Effect concentrations exceeding solubility of substance in test medium: No
Reported statistics and error estimates:
An experimental NOEC could not be determined due to significant reproductive effect at all concentrations tested (see Table 3), nor could a dose-response effect be observed at 0.13, 0.29, and 0.51 mg/L mean concentration. LOEC was 0.13 mg/L. Therefore, the NOEC for reproduction was calculated as one-half of the LOEC.

The 21-day NOEC for immobilization was 1.06 mg/L based on mean measured concentration.The 21-day LOEC values for both test organism reproduction and immobilization, as well as 21-day NOEC for immobilization, were based on t-statistics (p ≤ 0.05) and were determined using TOXSTAT (University of Wyoming 1989) statistical software.

Any other information on results incl. tables

Table 1, Initial and final test substance concentrations corrected for recovery
Test substance nominal concentration Replicate Measured concentration at various time points Mean measured concentration1
1st 2nd 3rd 4th
   
Initial concentration Day 2 Day 8 Day 15 Day 20  
   
0.0 (control) 0 0 0 0 0
0.25 0.19 0.02 0.14 0.27 0.20 ± 0.666
0.5 0.42 0.04 0.36 0.64 0.47 ± 0.151
1 0.79 0.08 0.80 0.96 0.85 ± 0.095
2 1.68 0.24 1.56 2.13 1.79 ± 0.301
4 3.61 1.04 3.00 4.76 3.79 ± 0.894
   
Final concentration Day 3 Day 9 Day 16 Day 21  
             
0.0 (control) A < 0.04 < 0.04 < 0.04 < 0.04  
  B < 0.04 < 0.04 < 0.04 < 0.04  
  C < 0.04 < 0.04 < 0.04 < 0.04  
  D < 0.04 < 0.04 < 0.04 < 0.04  
  Mean < 0.04 < 0.04 < 0.04 < 0.04 < 0.04
0.25 A 0.04 0.05 0.06 0.11  
  B 0.1 0.09 0.05 0.09  
  C < 0.04 < 0.04 0.07 < 0.04  
  D < 0.04 0.07 0.06 0.07  
  Mean 0.032 0.05 0.06 0.07 0.06 ± 0.013
0.5 A 0.12 0.07 0.08 0.10  
  B 0.19 0.10 0.08 0.09  
  C 0.10 0.11 0.05 0.06  
  D 0.11 0.10 0.09 0.10  
  Mean 0.13 0.10 0.08 0.09 0.10 ± 0.022
1 A 0.20 0.15 0.18 0.14  
  B 0.22 0.17 0.16 0.10  
  C 0.24 0.25 0.15 0.11  
  D 0.23 0.13 0.20 0.09  
  Mean 0.22 0.18 0.17 0.11 0.17 ± 0.055
2 A 0.74 0.40 0.37 0.13  
  B 0.39 0.34 0.28 0.17  
  C 0.35 0.59 0.39 0.13  
  D 0.47 0.36 0.34 0.17  
  Mean 0.49 0.42 0.35 0.14 0.33 ± 0.176
4 A 1.37 0.27 1.11 0.60  
  B 1.08 0.86 1.17 0.61  
  C 1.63 0.87 0.89, 1.183 ND4  
  D 1.49 0.93 0.74 0.56  
  Mean 1.39 0.73 1.02 0.59 1.00 ± 0.400
1, Mean concentrations for Day 21. Values were calculated excluding 2nd time point. Initial concentrations were low (Day 8) and in several cases less than the mean of the equivalent final concentrations (Day 9). Day 9 final concentrations were comparable to other final concentrations, but were excluded to avoid skewing calculated means.
2, Mean recalculated for this study summary using techniques in study report
3, Duplicate measures
4, ND, not determined because of complete mortality

Table 2, Mean test substance concentrations used for Days 14 and 21 endpoint calculations, corrected for recovery
Test substance nominal concentration (mg/L) Concentration used for endpoint calculation (mg/L)
Day 141 Day 212
0.0 (control) 0 0
0.25 0.11 0.13
0.5 0.28 0.29
1 0.51 0.51
2 1.09 1.06
4 2.50 2.40
1, Concentration calculated as average for Days 2 and 3
2, Concentration calculated as average for Days 2, 3, 15, 16, 20 and 21.

Table 3, Number of live offspring produced per day, cross-tabulated with nominal test substance concentation and replicate
Test day Number of live offspring produced
0.0 (Control) 0.25 mg/L 0.5 mg/L 1 mg/L 2 mg/L 4 mg/L
A B C D A B C D A B C D A B C D A B C D A B C D
7 50 37 0 0 15 48 0 7 19 15 10 33 31 34 48 36 0 0 0 0 0 0 0 0
8 0 26 10 0 30 9 1 23 19 31 63 33 95 51 39 34 0 13 51 0 0 0 0 0
9 49 33 85 114 47 16 86 59 31 40 24 45 0 39 0 26 74 14 4 39 0 0 0 0
10 172 150 10 13 38 125 12 31 73 110 28 99 110 95 170 146 58 40 5 28 0 0 0 0
11 121 120 107 157 20 24 123 12 56 36 148 31 92 68 31 53 4 17 80 0 1 0 0 0
12 35 41 127 70 0 7 53 1 2 9 35 76 16 81 0 56 247 88 17 159 0 0 0 0
13 152 176 1 0 16 192 3 19 183 117 95 151 213 200 212 259 0 69 2 23 0 0 0 0
14 59 166 198 332 208 3 265 235 88 50 219 83 52 45 31 9 0 7 81 0 0 0 0 0
14-day subtotal 638 749 538 686 374 424 543 387 471 408 622 551 609 613 531 619 383 248 240 249 1 0 0 0
14-day mean/replicate 652.8 432.0 513.0 593.0 280.0 0.3
14-day mean/original female 66.9 43.2 52.6 59.3 28.0 <1
15 85 16 217 42 0 37 93 86 3 75 22 112 0 98 0 78 198 54 21 154 0 0 0 0
16 231 223 0 0 117 344 0 1 88 151 93 160 123 109 205 102 16 124 11 47 0 0 0 0
17 75 146 141 379 292 25 269 245 179 109 268 104 214 153 103 138 0 30 101 0 0 0 0 0
18 69 24 238 45 1 39 126 146 76 61 5 113 0 105 0 72 117 18 71 20 0 0 0 0
19 293 271 15 2 88 427 14 15 112 135 112 164 110 110 180 92 233 168 14 154 0 0 0 0
20 58 112 109 359 317 5 162 192 252 197 300 161 308 233 209 198 0 80 79 0 0 0 0 0
21 114 101 291 74 27 50 263 236 113 93 0 94 0 133 0 90 32 2 132 0 0 0 0 0
21-day total 1563 1642 1539 1587 1216 1351 1470 1308 1294 1229 1422 1459 1364 1554 1228 1389 979 724 669 624 1 0 0 0
21-day mean/replicate1 1585.3 1336.3 1351.0 1383.8 749.0 <1
21-day mean/original female 162.6 133.6 138.6 138.4 74.9 <1
% reduction from control 17.8 14.8 14.9 53.9 <1

1, Number of live offspring produced per replicate were significantly reduced v. controls,p< 0.05

Table 4, Number of dead offspring produced per day, cross-tabulated with nominal test substance concentation and replicate
Test day Number of live offspring produced
0.0 (Control) 0.25 mg/L 0.5 mg/L 1 mg/L 2 mg/L 4 mg/L
A B C D A B C D A B C D A B C D A B C D A B C D
7 3 2 0 0 0 3 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
8 0 4 0 0 0 0 0 1 13 8 0 8 7 0 0 0 0 0 8 0 0 0 0 0
9 2 3 6 9 8 0 5 5 1 2 3 0 0 0 0 2 8 8 4 18 0 0 0 0
10 4 8 0 0 17 41 0 2 15 16 2 9 5 6 18 14 5 14 3 10 0 0 0 0
11 2 0 0 0 160 1 27 72 33 36 18 12 13 14 7 8 2 17 68 1 7 0 6 1
12 1 0 0 3 5 5 31 8 4 13 17 45 4 12 0 3 34 22 11 24 0 0 0 0
13 76 39 0 0 63 171 0 21 55 110 4 29 38 16 80 38 0 16 0 0 0 0 0 0
14 1 0 0 12 52 0 0 11 1 21 6 4 12 3 2 0 0 7 49 0 0 0 0 0
14-day subtotal 89 56 6 24 305 221 63 120 124 206 50 107 79 51 107 65 49 84 143 53 7 0 6 1
             
15 0 0 0 0 0 0 0 0 0 0 0 0 0 1 2 0 80 1 0 39 0 0 0 0
16 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 32 1 5 0 0 0 0
17 2 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 27 28 0 0 0 0 0
18 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1 6 0 9 17 0 0 0 0
19 0 0 0 0 0 0 0 0 0 0 0 0 0 0 7 1 4 9 3 32 0 0 0 0
20 0 0 0 0 0 0 0 0 0 0 0 2 0 0 1 0 0 18 6 0 0 0 0 0
21 0 0 0 0 0 0 0 0 11 0 0 0 0 0 0 0 6 0 26 0 0 0 0 0
21-day total 92 56 6 24 305 221 64 120 135 206 50 109 79 52 120 67 145 171 216 146 7 0 6 1

Table 5, Immobilization of D. magna in the chronic toxicity experiment.
Test substance nominal concentration (mg/L) Cumulative number of immobilized organisms (percent immobilized)1
Day 1 Day 2 Day 4 Day 7 Day 14 Day 21
0.0 (control) 0 (0) 0 (0) 0 (0) 1 (3) 1 (3) 2 (5)
0.25 0 (0) 0 (0) 0 (0) 0 (0) 2 (5) 2 (5)
0.5 2 (5) 2 (5) 2 (5) 3 (8) 3 (8) 3 (8)
1 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 4 (10)
2 1 (3) 1 (3) 1 (3) 1 (3) 1 (3) 3 (8)
4 0 (0) 1 (3) 5 (13) 18 (45) 27 (68)2 36 (90)2
1, percent immobilization is tabulated in paranthesis
2, Value significantly different from the control value atp≤ 0.05.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The 21-day NOEC for reproduction of Daphnia magna was 0.065 mg/L based on half of the LOEC, as determined using average measured concentration.
Executive summary:

The chronic toxicity of the test substance (CAS# 29590-42-9) to reproduction of aquatic invertebrates was assessed in 21-day assay based on OECD TG202 as promulgated for 1984. Test organisms less than 24 hours old at the beginning of the study. Medium was renewed daily. Paired concentration measurements, initial and final, were collected on Days 2 & 3, 15 &16, and 20 & 21. Mean measured concentrations were < 0.04 (control), 0.13, 0.29, 0.51, 1.06, 2.40 mg/L. Broods were first observed on Day 7, and live and dead offspring were counted daily until Day 21. Statistically significant effects on reproduction were observed for all concentrations tested. The NOEC was therefore estimated by taking half of the LOEC. The LOEC was 0.13 mg/L. The 21-day NOEC for reproduction of Daphnia magna was therefore 0.065 mg/L.

This study was performed in accord with then-current OECD test guidelines and GLP criteria. However, study conditions were not stably maintained, some analytical measurements were questionable, and substantial amounts of test substance were lost between renewals. Therefore, this study is classified as reliable with restrictions.