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Long-term toxicity to aquatic invertebrates

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Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-06-10 to 2005-10-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted in accordance to OECD guideline 211, with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
to allow aeration of exposure media.
Principles of method if other than guideline:
Due to unavoidable biodegradation processes, which have to be accepted as an intrinsic and realistic feature of the test procedure, sever oxygen depletion occurred. In contradiction to the test guideline, cautious aeration with sterile filtered air was implemented to overcome this.
GLP compliance:
yes (incl. certificate)
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. 
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 discarded. 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. The test media were not stored for more than 1 - 2 hours prior to testing.

- Controls: the controls were prepared using test media only.

- Evidence of undissolved material: the test solutions of fatty alcohols were not filtered as it was shown that the filtation procedure caused highly variable and much lower test concentrations than the water solubility predicted from physico-chemical models. When omitting the filtration procedure, the concentrations were clearly higher than the solubility predicted by models and there was no clear indication of a trend to saturation. It was therefore concluded that the measured concentrations comprised a fraction of dissolved test substance and a fraction of finely dispersed material. It was considered technically impossible to avoid the presence of very fine undissolved particles or droplets at the higher test concentrations.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM

- Common name: freshwater water flea

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

- Age of parental stock: adult daphnia at least 3 weeks old are separated from the the stock population by sieving. The test animals used in the test were 0-24h old.

- Feeding during test

- Food type: suspensions of unicellular green alga

- Amount: The content of food in the test suspensions, measured as turbidity at 758 nm, increased during the test from 7 mg C/L equivalents to 15 mg C/L equivalents.

- Frequency: fed at each water renewal.


ACCLIMATION

- Acclimation period: 1 week

- Acclimation conditions: batches of 30 to 50 animals are held at room temperature in ca. 1800 L dilution water. The water is changed once a week.

- Type and amount of food: During the week animals are fed with an algal suspension (Desmodesmus subspicatus) and LiquizellR (HOBBY) Algae growing in the log phase are centrifuged and the pellet is re-suspended in a few ml of medium. 30 ml of this suspension is given to 1L daphnia medium.

- Feeding frequency: daily

- Health during acclimation: good.


METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: newborn daphnia were separated by sieving, the first generation being discarded.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
none
Hardness:
0.9 mmol/L
Test temperature:
range 21.0 to 22.0°C
pH:
range: 9.3 to 9.5 
Dissolved oxygen:
The oxygen saturation never fell below 56 % (4.0 mg/L).
Salinity:
N.A.
Nominal and measured concentrations:
Nominal: 0, 25, 69, 185 and 500 ug test item/L
Measured concentrations: test solutions were The geometric means of mean measured initial and aged concentrations after 24 h hours were 
Details on test conditions:
TEST SYSTEM

- Test vessel: glass flask

- Type: closed with autoclaved silicone stoppers.

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

- Aeration: yes. 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.

- Renewal rate of test solution: daily

- No. of organisms per vessel: 1

- No. of vessels per concentration : 10

- No. of vessels per control : 10


TEST MEDIUM / WATER PARAMETERS

- Source/preparation of dilution water: purified drinking water.

- Metals: Cu 0.0031 mg/L, Fe 0.0066 mg/L, Mn 0.0003 mg/L, Zn 0.0075 mg/L

- Alkalinity: 1.6 mmol/L

- Ca/mg ratio: Ca 0.8 mmol/L, Mg 0.1 mmol/L

- Conductivity: 202 uS/cm

- Intervals of water quality measurement:


OTHER TEST CONDITIONS

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

- Light intensity: in the range 585 to 647 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: - Mortality (immobility) of parental generation Daphnia magna - Age at first brood - Total number of offspring per replicate - Cumulative Number of live offspring per surviving female at the time of recording - Intrinsic rate of increase, r - Individual length of adults


RANGE-FINDING STUDY
The range finding studies used to determine test concentration are referenced as:
Schafers C (2005a): Pre-studies with dodecanol (C12 OH) for the preparation of a study plan for a Daphnia magna reproduction test (OECD 211). Fraunhofer IME, Schmallenberg, Germany.
Schafers C (2005a): Pre-studies with pentadecanol (C15 OH) for the preparation of a study plan for a Daphnia magna reproduction test (OECD 211). Fraunhofer IME, Schmallenberg, Germany.
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
14 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: cumulative number of offspring
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
95 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
of parent Daphnia
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
13 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: cumulative number of offspring
Duration:
21 d
Dose descriptor:
other: EC20
Effect conc.:
34 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: cumulative number of offspring
Details on results:
- Mortality of parent animals: At the highest treatment level 6 out of 10 introduced animals died, 5 of them before reproduction, therefore the highest treatment was not considered in the evaluation of reproduction and growth.

- No. of offspring produced per day per female: for control, 3, 5, 14 and 95 µg/L was respectively: 3.2, 3.19, 3.07, 3.04 and 2.56 (see Table 1)

- Body length and weight of parent animals: The average growth length was (respective to nominal concentrations) control - 4.83 mm, 25 ug/L - 4.37 mm, 69 ug/L - 4.47 mm, 185 ug/L - 4.38 mm, 500 ug/L - 4.76 mm (see Table 1)

- Time to first brood release or time to hatch: the mean age at first brood was between 8.1 and 8.4 days for all relevant treatment levels (see Table 1)

- Brood size: for control, 3, 5, 14 and 95 µg/L the cumulative number of offspring per female was 68, 68, 63, 61 and 64 respectively (see Table 1)
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 1. Survival, growth and reproduction data.

 Test item nominal conc. (¿g/L)  Survival (%)  Growth (length) Mean ± SD (mm)  Age at first brood Mean ± SD (days)  Cumulative offspring per female Mean ± SD (#)  Age at first brood Mean ± SD (1/days)
Control  100  4.83 ± 0.35  8.2 ± 0.79  68.0 ± 8.9  0.320 ± 0.024
 25  100  4.37 ± 0.39  8.2 ± 0.92  68.0 ± 11.6  0.319 ± 0.026
 69  100  4.47 ± 0.31  8.4 ± 0.84  63.0 ± 9.8  0.307 ± 0.018
 185  100  4.38 ± 0.41  8.1 ± 0.99  61.0 ± 7.1  0.304 ± 0.019
 500  40  4.76 ± 0.40  8.6 ± 1.14  64.0 ± 19.3  0.256 ± 0.044


Table 2. Calculated statistics relative to initial concentrations.


 EC10

150 µg test item/L

 EC20

520 µg test item/L

 LOEC

530 µg test item/L

 NOEC

160 µg test item/L

Table 3. Nominal and measured concentrations.

 Nominal concentration (¿g/L)  Measured concentration - FRESH media (¿g/L) (Mean (SD %))  Measured concentration - OLD media (¿g/L) (Mean (SD %))  Geometric mean (¿g/L)
 25  20 (25%)  <LOQ (39%)  3.4
 69  56 (21%)  <LOQ (5%)  5.3
 185  163 (24%)  1.1 (100%)  13.6
 500  534 (19%)  17 (135%)  95.3


Validity criteria fulfilled:
yes
Conclusions:
A reliable 21 day NOECreprovalue of 14 ¿g/Lwas determined for the effects of the test substance on reproduction (cumulative number of offspring) of the freshwater test organism Daphnia magna.
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Principles of method if other than guideline:
Method: other: Comparable to OECD guideline 202, Part 2.
GLP compliance:
yes
Analytical monitoring:
yes
Test organisms (species):
Daphnia magna
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Basis for effect:
other: reproduction rate and mortality
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
3 mg/L

RESULTS:
Concentration (mg/L)     0     1    3     10    30    100
First day of offspring  9-12  9-12 9-12  9-12  9-12 9-12
Survival rate of         90    90   90    50    45    40
adults (%) 
Average number of young  106   112  84    55    25    28
animal per adult

Standard deviation       3.6   3.5  13.2  15.3  12.6  4.3
Significant (p<0.05)     --    no   yes   yes   yes   yes

NOEC = 1 mg/L
LOEC = 3 mg/L

CONTROL SUBSTANCE: No use of control substance reported.

Description of key information

Long-term toxicity to aquatic invertebrates: 21 day EC10 0.013 mg/l (measured) in accordance with test guideline OECD 211 (Daphnia magna).

Key value for chemical safety assessment

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

Additional information

A reliable 21 day EC10 value of 0.013 mg/l (geometric mean measured concentration) has been determined for the effects of dodecanol (CAS 112-53-8) on reproduction (cumulative number of offspring) of the freshwater test organism Daphnia magna, in accordance with guideline OECD 211. The study reflects the lowest reliable value that is available for this endpoint and has been selected as key.

This value is supported by a second long-term invertebrate study which reports a 21 day NOEC of 1 mg/l for the effects of dodecanol on reproduction rate and mortality of Daphnia magna (no EC10 was reported) [Henkel KGaA, 1992).

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.

 

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.