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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:
July 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted in accordance with GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
to allow aeration of exposure media.
Principles of method if other than guideline:
Deviation: As a deviation from OECD Guideline 211, all test vessels were aerated with sterile filtrated synthetic air: the autoclaved silicone stoppers were fitted with fine glass capillaries connected to the aeration unit. The aeration was necessary to avoid severe oxygen depletion due to the increase of transferred bacteria with growing Daphnia magna as observed in pre-studies and the associated oxygen consumption by the degradation of the test substance
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All the test concentrations were sampled for chemical analysis three times a week at renewal of the test media. 

- Sampling method: A 500 mL aliquot of the fresh solutions was used for analysis. After 24 h, at the next renewal, the aged test liquids were pooled (vessels 1- 5 and  6-10) and analysed. 

- Sample storage conditions before analysis: The test media were not stored for more than 1 - 2 hours prior to testing.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: Test solutions were prepared daily by stirring  the test substance in test media under slow stir conditions (21 h) in sterilized mixing vessels. The mixing vessels were cylindrical brown glass bottles with teflon covered screw caps, fitted with a drain port near the bottom for drawing off the test solution. The volume of the mixing vessels was 2 L. After stirring, the contents of the vessels were left to settle for 2 h. The saturated aqueous phase was then taken out of  the drain port. The first fraction 0-100 mL was withdrawn. The fraction between 100 and 1800 mL was used for rinsing (200 mL) and filling (1000 mL) the test flasks for toxicity testing and for analytical measurements (500 mL), if done. Rinsing of the test vessels was carried out to saturate the surfaces of the test vessels. After filling, the vessels were closed immediately by using autoclaved silicone stoppers and only opened to introduce the test organisms and again at the renewals of the test media. 

- Evidence of undissolved material: The test solution was not filtered as filtration was shown to cause highly variable and much lower test concentrations the water solubility predicted. When omitting the filtration process, the concentrations were clearly higher than the solubility predicted.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM

- Common name: freshwater flea

- Source: Umweltbundesamt (German Federal Environment Agency). Test organisms bred in the laboratory of the Fh-IME (testing facility).

- Age of parental stock: 4-24 hrs.

- Feeding during test

- Food type: suspension of D. subspicatus.

- Frequency: at each test renewal


ACCLIMATION

- Acclimation conditions: renewal of waters in the cultures is weekly, not daily.

- Type and amount of food: Desmodesmus subspicatus and LiquizellR (HOBBY)

- Feeding frequency: daily


METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: newborn daphnia were separated by sieving, the first generation was discarded.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
Not reported
Test temperature:
range: 20.0 to 21.0°C
pH:
range: 9.4-9.5
Dissolved oxygen:
oxygen saturation never fell below 70 % (5.7 mg/L)
Nominal and measured concentrations:
Nominal test concentrations were 0, 152, 411, 1110 and 3000 µg test item/L. Initial mean measured concentrations of freshly prepared test solutions were 1.6, 122, 351, 962, 2800 µg/L.
Geometric means of mean measured initial and aged concentrations after 24 h hours were See Table 3 for further details.
Details on test conditions:
TEST SYSTEM

- Test vessel: numbered vessel flask

- Type: closed, with autoclaved silicone stoppers.

- Material, size, headspace, fill volume: glass flasks, filled with 100 ml test solution.

- Aeration: yes, with sterile filtrated synthetic air through glass capillaries.

- Renewal rate of test solution (frequency):

- No. of organisms per vessel: 10

- No. of vessels per concentration: 1

- No. of vessels per control: 1


TEST MEDIUM / WATER PARAMETERS

- Source/preparation of dilution water: purified drinking water, autoclaved before use. The purification included filtration with activated charcoal, passage through a limestone column and aeration until oxygen saturation.

- Total organic carbon: DOC 1.72 mg/L

- Metals: Ca 0.6 mmol/L, Mg 0.1 mmol/L, Nitrate <0.5 mmol/L. Nitrite <0.002 mg/L, Cu 0.0031 mg/L, phosphate 0.56 mg/L, Fe 0.0066 mg/L, Mn 0.0003 mg/L, Zn 0.0075 mg/L

- Alkalinity: 1.0 mmol/L

- Ca/mg ratio: 6:1

- Conductivity: 227 uS/cm

- Intervals of water quality measurement: daily


OTHER TEST CONDITIONS

- Photoperiod: day/light cycle = 16/8 hours

- Light intensity: 588 to 657 lux


EFFECT PARAMETERS MEASURED: The parent Daphnia magna were assessed visually daily for immobility and any other abnormalities in appearance 
and behaviour. At study termination, the length of the adults was measured by digital photography and image analysis and their statistics compared with those of the control animals. The newborn Daphnia magna in each beaker were counted at each daily renewal of the test solutions, inspected for abnormalities in condition, and removed. The following endpoints observed in the reproduction test were evaluated quantitatively:
o Mortality (immobility) of parental generation Daphnia magna;
o Age at first brood;
o Total number of offspring per replicate;
o Cumulative Number of live offspring per surviving female at the time of recording;
o Intrinsic rate of increase, r;
o Individual length of adults.

RANGE-FINDING STUDY
- Results used to determine the conditions for the definitive study: acute toxicity test D. magna LC50 of 2300 ug/L
Reference substance (positive control):
not specified
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
210 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: reproduction rate
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
110 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: reproduction rate
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
370 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: reproduction rate
Duration:
21 d
Dose descriptor:
other: EC20
Effect conc.:
670 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: reproduction rate
Details on results:
- Mortality of parent animals: during the test no parental mortality occured, neither were any clinical signs observed, except for the highest treatment level where all animals died before reproduction.

- No. of offspring produced per day per female: see Table 1 for details.

- Body length and weight of parent animals: see Table 1 for details.

- Time to first brood release or time to hatch: see Table 1 for details.

- Brood size: see Table 1 for details.
Reported statistics and error estimates:
The evaluation of the concentration-effect-relationships and the calculations of effect concentrations were based on mean measured initial concentrations as multiple peak concentrations, as well as on geometric means between mean measured initial and aged (24h) test concentrations. For each endpoint, the NOEC, LOEC, and, if possible, the EC50, EC20 and EC10 were determined. A LOEC and NOEC were calculated by ANOVA followed by Williams' test or an appropriate non-parametric test suggested by the ToxRat program. When the test results showed a concentration-response relationship, the data were analysed by regression using Probit-analysis assuming log-normal distribution of the values using the computer program ToxRat program.

Table 1a. Survival, growth and age at first brood data (21 day).

 Test item Nominal Conc. (μg/L)  Survival Mean ± SD

(%)

 Growth (length) Mean ± SD

(mm)

Age at first brood Mean ± SD  

(days)

 Control  100

5.41 ± 0.22

8.9 ± 0.74

 152  100

5.52 ± 0.19  

9.2 ± 0.79

 411  100

5.43 ± 0.21

9.0 ± 0.82

 1,110  100

5.26 ± 0.38

9.4 ± 0.97

 3,000  0  n.d.  n.d.

Table 1b. 21 day Reproduction data.

 Test item nominal conc. (μg/L)  Cumulative offspring per female Mean ± SD (#)

 Intrinsic rate of increase r  Mean ± SD

(1/d)

 Control

68.1 ± 9.5  

0.294 ± 0.017

 152

68.0 ± 5.6  

0.297 ± 0.018

 411

62.9 ± 5.8

0.281 ± 0.011

 1,110

58.6 ± 7.7 * 

0.277 ± 0.024 *

 3,000  n.d.  n.d.




* significant difference to control according to Williams-test (a = 0.05,  one-sided smaller)


Table 2. Calculated statistics relative to offspring production rate, related to daily initial concentrations.

 EC10

610 µg test item/L

 EC20

1500 µg test item/L

 LOEC

960 µg test item/L

 NOEC

350 µg test item/L

Table 3. Results of analytical monitoring of the test substance. The Fresh and Old measurements are the arithmetic mean of three weekly measurements, while the Geometric mean is the geometric mean of fresh and old measurements.

 Nominal concentration (μg/L)  Fresh (Mean (SD %)) (μg/L)  Old (Mean (SD%)) (μg/L)  Geometric Mean (μg/L)
 152  122 (15%)  4.4 (119%)  23.3
 411  351 (10%)  33 (109%)  107
 1110  962 (30%)  140 (91%)  367
 3000  2800 (10%)  537 (65%)  1227
Validity criteria fulfilled:
yes
Conclusions:
A 21 d EC10 value of 210 µg/l and NOEC value of 110 µg/L have been determined for the effects of 1-Decanol on the reproduction rate of Daphnia magna.
Executive summary:

The data was obtained generally in accordance with standard test guideline OECD 211. However some modifications to the normal guideline procedures were necessary to reduce losses of test substances due to the extensive and rapid biodegradation of the LCAAs. Gentle aeration of test vessels was required as degradative losses of LCAAs resulted in unacceptably low dissolved oxygen concentrations. Algae have been found to metabolize LCAAs and this is an unavoidable occurrence in long-term studies with Daphnia magna fed with algae. No modifications could be made to counter this without conducting further research into an alternative diet.

Description of key information

Long-term toxicity to invertebrates: 21-d EC10 210 µg/l and NOEC 110 µg/L (OECD 211 with adaptations for rapid degradation of test substance)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
210 µg/L

Additional information

A 21-d EC10 value of 210 µg/l and NOEC value of 110 µg/L have been determined for the effects of decanol on the reproduction of Daphnia magna. The study used adapted methodology necessary to minimise the extensive losses of test substance due to biodegradation in the test system, and is considered definitive for this endpoint.

Discussion of trends in the Category of C6-24 linear and essentially-linear aliphatic alcohols:

Linear LCAAs

Data of an acceptable quality are available for 21-day reproduction studies with Daphnia magna for the single carbon chain length LCAAs 1-octanol (Kuhn et al., 1989), 1-decanol, 1-dodecanol, 1-tetradecanol, 1-pentadecanol (Fraunhofer Institute, 2005a-d respectively), pentadecanol branched (ABC 1999a) and octadecanol branched (ABC 1999c). The data were obtained generally in accordance with standard test guideline OECD 211. However some modifications to the normal guideline procedures were necessary to reduce losses of test substances due to the extensive and rapid biodegradation of the LCAAs. The following changes to typical protocols were therefore adopted to enable the performance of high-quality and meaningful studies:

 

Vessels were closed, to reduce entry of bacteria from the atmosphere;

Gentle aeration of test vessels was required as degradative losses of LCAAs resulted in unacceptably low dissolved oxygen concentrations;

Test solution renewals were made daily, with confirmatory analysis on both renewed and initial test solutions;

Static renewal was determined to be the best exposure regime for long chain aliphatic alcohols as this reduced the transfer of LCAAs -degrading or consuming microbes (as compared to flow-through systems, where it becomes increasingly difficult to discourage acclimation and bio film formation; see Brixham Environmental Laboratory, AstraZeneca, 2004);

Saturated alcohol stock solutions were prepared daily for each test concentration. This involved a detailed preparatory method to reduce the possibility of insoluble material being present in the tests (Fraunhofer Institute, 2005a, b);

Daphnia magna were carefully rinsed with each daily transfer to reduce bacterial cross over to fresh exposure solutions. As Daphnia magna grow in size, this becomes less effective; and,

Dilution water and test vessels were autoclaved prior to use each time (Fraunhofer Institute, 2005a, b, c, d).

 

Algae have been found to metabolize LCAAs and this is an unavoidable occurrence in long-term studies with Daphnia magna fed with algae. No modifications could be made to counter this without conducting further research into an alternative diet.

 

In spite of the guideline modifications significant losses of test substance still occurred. It was therefore necessary to report the results both in terms of the mean of the measured concentrations in the fresh media and the mean of the measured concentrations in the fresh and old media. The test substance renewal interval was 24 hours. Survival and reproduction endpoints have been summarised using standard statistical techniques. Conclusions for each test are presented as both NOEC and EC10. The 1-octanol and 1-octadecanol study are reliability 2, valid with restrictions; the other studies are reliability 1.

 

The effect of LCAAs on Daphnia magna survival is generally less sensitive than the effect on reproduction. A pattern of increased toxicity with increasing chain length is also apparent. In the octanol study, the most sensitive and only reported effect was on time to first brood release which occurred at 1000 µg/L (nominal concentration). For comparison of results across chain lengths and structure activity models the response for survival and reproduction was assumed to be equal to the effect on time to first brood.

 

The data indicates that for survival and reproduction, the NOEC and EC10 values increase from C14 to C15. The study authors report that this is almost certainly due to exceeding the limit of water solubility as would be expected from conventional toxicological theory (Rufli et al., 1998). Under these circumstances a more accurate interpretation of the results might be obtained by setting the exposure to the solubility of the substance (i.e. 49 µg/L). This has the effect of lowering the toxicity values but they are still higher than those for the C14 substance. This pattern is not in keeping with the trend of reducing short-term toxicity values (i.e. higher toxicity) observed between the C8 and C14 alcohols. Similarly, the NOEC identified for C18 is a limit value of >980 µg/L but a lower value would have been obtained if a lower loading had been tested. A more accurate NOEC would therefore be obtained by expressing it as greater than the water solubility of the test substance, which is 10 µg/L.

It must be appreciated that significant uncertainty exists in identifying the true exposure concentrations in the region of the water solubility of a substance. The water solubility values of the LCAAs category decrease with increasing chain length (see section 1.4 for further details.). In a review of aquatic toxicity testing of sparingly soluble compounds Rufli et al. (1998) point out that interpretation of toxicity responses observed above the solubility limit is aggravated by artefacts and that testing should only occur at or below the limit. For LCAAs with carbon numbers greater than C15 there are significant experimental difficulties in producing, maintaining and quantifying exposures of the test substance due to progressively lower solubility, while exceptionally rapid biodegradability would remain unchanged. This explains why there are no data for such substances. 

 

However, based on the trends observed in the available data, it is expected that for linear LCAAs with carbon numbers >C15 the NOEC and EC10 for long-term effects on mortality and reproduction would be above the solubility limit (Schäfers et al., 2009).

 

 

Multi-constituent LCAAs

No measured data are available for multi-constituent substances of different carbon chain length LCAAs.

 

References:

ABC Laboratories (1999a). Chronic toxicity of PMN P98-960 during the complete life-cycle of Daphnia magna under flow-through test conditions. ABC Study No. 45414. Oct.13 1999. ABC Laboratories Inc. 7200 E. ABC Lane, Columbia, Missouri 65202.

 

ABC Laboratories (1999c). Chronic toxicity of PMN P98-963 during the complete life-cycle of Daphnia magna under flow-through test conditions. ABC Study No.45658. Oct.13 1999. ABC Laboratories Inc. 7200 E. ABC Lane, Columbia, Missouri 65202.

 

Kuhn, R., Pattard, M., Pernak, K., and Winter, A. (1989). Results of the harmful effects of water pollutants to Daphnia magna in the 21 day reproduction test.  Wat. Res. 23(4): 501-510.

 

Rufli, H., P. R. Fisk, A. E. Girling, J. M. H. King, R. Lange, X. Lejeune, N. Stelter, C. Stevens, P. Suteau, J. Tapp, J. Thus, D. J. Versteeg, H. J. Niessen. 1998. Aquatic toxicity of sparingly soluble, volatile, and unstable substances and interpretation and use of data. Ecotoxicology and Environmental Safety 39 (2):72-77.

 

Fraunhofer Institute, 2005a. Daphnia magna, reproduction test in closed vessels following OECD 211. C10 fatty alcohol. GLP code: SDA-005/4-21. Fraunhofer Institute for Molecular Biology and Applied Ecology (IME) 57377 Schmallenberg, Germany.

 

Fraunhofer Institute, 2005b. Daphnia magna, reproduction test in closed vessels following OECD 211. C12 fatty alcohol. GLP code: SDA-001/4-21. Fraunhofer Institute for Molecular Biology and Applied Ecology (IME) 57377 Schmallenberg, Germany.

 

Fraunhofer Institute, 2005c. Daphnia magna, reproduction test in closed vessels following OECD 211. C14 fatty alcohol. GLP code: SDA-006/4-21. Fraunhofer Institute for Molecular Biology and Applied Ecology (IME) 57377 Schmallenberg, Germany.

 

Fraunhofer Institute, 2005d. Daphnia magna, reproduction test in closed vessels following OECD 211. C15 fatty alcohol. GLP code: SDA-002/4-21. Fraunhofer Institute for Molecular Biology and Applied Ecology (IME) 57377 Schmallenberg, Germany.

 

Schäfers, C. Boshof, U. Jürling, H. Belanger, S.E. Sanderson, H. Dyer, S.D. Nielsen, A.M. Willing, A. Gamon, K. Kasai, Y. Eadsforth, C.V. Fisk, P.R. Girling, A.E., 2009. Environmental properties of long chain aliphatic alcohols. Part 2: Structure-activity relationship for chronic aquatic toxicity of long-chain alcohols. Ecotoxicology and environmental safety. 72(4): 996-1005.