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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:
2005-08-22 to 2005-09-31
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP.
according to
OECD Guideline 211 (Daphnia magna Reproduction Test)
to allow aeration of the vessels, test media eas prepared daily, water changes occured daily, daphnia were carefully rinsed at each water change.
Principles of method if other than guideline:
Deviations from the guideline was as follows: due to the unavoidable biodegradation process caused by the presence of algae (daphnia feed), severe oxygen depletion occurred, cautious aeration with sterile filtered air was implemented in order to overcome this.

GLP compliance:
yes (incl. certificate)
Analytical monitoring:
Details on sampling:
- Concentrations: Pooled samples were taken at each concentration from vessels 1-5 and 6-10 for the measurement of the aged test solution.

- Sampling method: Freshly prepared test solutions aliquots (500 ml), obtained before distribution to the replicates, and aged test solutions aliquots (500 ml), obtained before the renewal on the next day of all test concentrations, were sampled three times a week.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

- Method: The test solutions were prepared by stirring the test substance in test media under slow stir conditions (1 h) in sterilised 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 2L. The loading of the mixing vessels corresponded to 24.4, 68.6, 185.2 and 500 ug/L and was targeted at achieving an initial top concentration of approximately 350 ug/L, taking into account 30% adsorption to the glassware as shown in pre-studies. For each concentration, an ethanoic stock solution was prepared. 1 ml of the appropriate ethanoic stock solution was pipetted into the 2 L mixing vessel taken from the sterilisation process, the still warm mixing vessel slowly turned to cover maximum area of the glass walls with ethanoic solution. The movements were continued until the liquid was no longer visible. The ethanol solution did not moisten the inner area of the flask where a sharp magnetic stirrer was later placed. After the addition of the stirring bar the vessels were sealed leaving only a small headspace. The contents of the vessels were stirred at 100 rpm for approximately 21 h. After stirring the contents of the vessels were left to settle for 2 h. The aqueous saturated 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 vessels was carried out to saturate the surfaces of the vessels. The test media were not stored for more than 1-2 h prior to testing.

- Controls: The control medium was prepared similarly as above with 1 ml of ethanol

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): ethanol

- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): <1ml

- Evidence of undissolved material (e.g. precipitate, surface film, etc): the test solutions of fatty alcohols were not filtered as the filtration procedure was shown to cause highly variable concentrations of alcohols, and lower than the ones predicted by the physico-chemical model. When omitting the filtration procedure, the concentrations were clearly higher than the predicted solubility. This is thought by the authors of the report to be due to the fact that the measured concentrations comprised a fraction of dissolved test substance and a fraction of finely dispersed material. It was considered to be technically impossible to avoid the presence of very fine undissolved particles or droplets at the highest concentration.
Test organisms (species):
Daphnia magna
Details on test organisms:

- Common name: freshwater flea or daphnia

- Source: Umweltbundesamt (German Federal Environment Agency)

- Age of parental stock (mean and range, SD): 4-24h old

- Feeding during test

- Food type: suspensions of unicellular green algae. The suspensions of Desmodesmus subspicatus ere analysed for microbial contamination. The content food in the test suspensions, measured as turbidity at 758 nmm, increased during the test from 7 mg C/L equivalents to 15 mg C/L equivalents.
- Frequency: daily, with the change of test solution.


- Acclimation period: the organisms were bred at the testing facility. Adult daphnia (>3 weeks old) were separated from the stock population, and kept in batches of 30-50 animals in 1800 ml dilution water at room temperature for a week. Newborn daphnia were separated by sieving, the first generation was discarded.

- Acclimation conditions (same as test or not): same though kept at room temperature (temperature not recorded).

- Type and amount of food: algal suspension (Desmodesmus subspicatus) and LiquizellR (HOBBY). Algae growing in the log phase were centrifuged and re-suspended in a few ml of medium. 30 ml of this suspension was given to the 1L of daphnia medium.

- Feeding frequency: daily

Test type:
Water media type:
Limit test:
Total exposure duration:
21 d
Post exposure observation period:
0.7 mmol/L
Test temperature:
21 +/- 1 deg C
Dissolved oxygen:
>75% or >6 mg/L
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 0, 24.4, 68.6, 185.2, 500 ug test item/L.
Geometric means of mean measured initial and aged concentrations after 24h (effect concentration): 0, 1.6, 3.6, 13 and 77 ug test item/L.
Mean measured concentrations of fresh test solutions: Biodegradation caused the majority of the change in the fresh and aged test solution concentrations, followed by adsoprtion to the test vessels.
Details on test conditions:

- Test vessel: beakers

- Type (delete if not applicable): closed with an autoclaved silicone stopper

- Material, size, headspace, fill volume: glass beakers filled with 100 ml test medium

- Aeration: yes, with sterile filtrated synthetic air: the autoclaved silicone stoppers were fitted with fine glass capillaries connected to the aeration unit. The capillaries were introduced just below the surface of the test solutions.

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

- No. of organisms per vessel: 1

- No. of vessels per concentration (replicates): 10

- No. of vessels per control (replicates): 10


- Source/preparation of dilution water: purified drinking water (filtered and autoclaved before use).

- Total organic carbon: DOC 0.62 mg/L

- Metals: Cu 0.0032 mg/L; Fe 0.0138 mg/L; Mn 0.0007 mg/L and Zn 0.0045 mg/L

- Alkalinity: 1.7 mml/L

- Ca/mg ratio: Ca 0.6 mmolL, Mg 0.1 mmol/L; ratio = 6:1

- Conductivity: 214 uS/cm

- Culture medium different from test medium: no

- Intervals of water quality measurement: daily


- Photoperiod: light/dark cycle of 16/8 hours

- Light intensity: <1000 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : effects on growth (adult length at test termination), reproductive performance, immobility and any other abnormalities were investigated daily. The following endpoints were evaluated quantitavely:
- mortality (immobility) of parental generation of Daphnia magna
- age at first brood
- total number of offspring per replicate
- cumulative number of offspring per surviving female at the time of recording
- intrinsic rate of increase
- individual length of adults

VEHICLE CONTROL PERFORMED: ethanol was included in the control


- Test concentrations: up to 500 ug/L (nominal)

- Results used to determine the conditions for the definitive study: highest nominal concentration of 500 ug/L chosen in order to achieve the highest measured concentration near the maximum estimation of water solubility.
Reference substance (positive control):
21 d
Dose descriptor:
Effect conc.:
6.3 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: (cumulative number of offspring)
21 d
Dose descriptor:
other: EC20
Effect conc.:
23 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: (cumulative number of offspring)
21 d
Dose descriptor:
Effect conc.:
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: (cumulative number of offspring)
21 d
Dose descriptor:
Effect conc.:
1.6 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: (cumulative number of offspring)
Details on results:
- Mortality of parent animals: at the highest treatment 3 of the 10 introduced daphnia died between days 9 and 16.

- No. of offspring produced per day per female: for control, 24.4, 68.6, 185.2, 500 ug/L respectively: 3.7, 3.6, 3.5, 3.4 and 3.4

- Body length and weight of parent animals: adult body length showed no significant difference between treatments.

- Time to first brood: the mean first brood occurred between 7.7. and 8.2 days at all concentrations, no statistically significant deviation from controls.

- Brood size: the cumulative number of offspring was significantly reduced by 8, 15 and 20% at the three higher treatments. The intrinsic rate of increase (r) was significantly reduced at the two higher concentrations, but by less than 10%.

- Effect concentrations exceeding solubility of substance in test medium: the test solutions of fatty alcohols were not filtered and the resulting concentrations were clearly higher than the predicted solubility.
Reported statistics and error estimates:
The LOEC and NOEC were calculated by ANOVA followed by William's test or an appropriate non-parametric test suggested bt the ToxRat program. When the results showed a dose-response relationship, the data were analysed by regression using Probit-analysis assuming log-normal distribution of the values using the program ToxRat.

Table 1: Effect of 1 -tetradecanol on growth and survival of D. magna


Treatment (¿g/l)

Mortality of parents

Growth (length))

Age at first brood

Cumulative offspring per female

Intrinsic rate of increase r


Mean ± SD (mm)

Mean± SD (days)

Mean± SD

Mean ± SD (1/d)

Control *


4.47 ± 0.32

7.8± 0.8

86.6± 5.7

0.368± 0.0319



4.83± 0.31

7.7± 0.7

84.0± 7.4

0.364± 0.0343



4.50± 0.38

8.1± 0.7

79.7± 9.4

0.347± 0.0328



4.49± 0.27

8.2± 0.8

73.9± 8.5

0.338± 0.0347



4.73± 0.40

7.9± 0.6

70.4± 8.2

0.338± 0.0241


* thecontrol is also the solvent control (ethanol).

The daily transfer of daphnia, although rinsed thoroughly, did cause the re-introduction of bacteria, which increased in number with growth of carapace and resulted in more pronounced losses in the last two weeks of the reproduction studies.


Table 2: Nominal and measured concentrations for 21 days.


Measured concentration (¿g/L) results

Nominal concentration





 Fresh media

Mean* ±st. dev. (%)

 10 ± 49%

51 ± 21% 

138 ± 20% 

367 ± 31%

 Old media

Mean* ±st. dev. (%)



1.2 ± 134%

16 ± 157%


% of nominal (ref. to mean)





* arithmetic mean of 3 weekly measurements

¥ geometric mean of fresh and old means


Table 3. Effect concentrations relative to daily initial concentrations (based on cumulative number of offspring).


 ¿g test item/L











Validity criteria fulfilled:
A reliable 21 d NOECrepro value of 1.6 ug/L has been identified for the effect of the test substance on the cumulative number of offspring of D. magna.

Description of key information

Long-term toxicity to invertebrates: 21 d EC10: 6.3 µg/L (0.0063 mg/l) (mean measured concentrations) for the effects of the test substance on reproduction of Daphnia magna (guideline OECD 211).

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
0.006 mg/L

Additional information

A reliable 21 day EC10 value of 0.0063 mg/l (mean measured concentration) has been determined for the effects of tetradecanol (CAS 112 -72-1) on reproduction of the freshwater test organism Daphnia magna (Fraunhofer, 2005) The study reflects the only reliable value that is available for this endpoint and has been selected as key. During this test the report authors state, "It was considered to be technically impossible to avoid the presence of very fine undissolved particles or droplets at the highest concentration." The highest test concentration was 0.367 mg/l therefore the presence of undissolved material should not have affected the test organisms at the concentration at which the EC10 is reported.

The study also reports a NOEC of 0.0016 mg/l for effects on reproduction, which is recorded here for the purpose of setting M-factors for classification and labelling.

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. 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. This statement is supported by data on C15 and C15 branched, where the NOEC was not achieved at the solubility limit.


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 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.



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.