[No public or meaningful name is available]

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

No data available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study conducted according to sound scientific principles, no GLP or details on analytical monitoring conducted are reported.

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: Federal Environment Agency (1984)

Deviations:

not specified

Remarks:

Analyitcal monitoring sampling days were reduced to 2

GLP compliance:

not specified

Analytical monitoring:

yes

Details on sampling:

Sampling method: Samples were taken twice from selected concentration levels of the test series during the test period and analysed chemically. The first sampling was taken before the 7th day previous to any offspring appearance, the second sample was taken between day 16 and day 21. No further details on analytical methods were presented.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: the test substance was dispersed to make up a stock solution of 200 mg/L, then gradual serial dilutions (corresponding to a ratio of 1:2) of the stock solution were made to produce the concentration range tested.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain: IRCHA
- Age of parental stock: maintained in accordance with the procedure practised since 1978
- Feeding during test
- Food type: Tetramin-Haupfutter (fish feed) and activated sludge.

ACCLIMATION
- Type and amount of food: dry algae Scenedsmus spp. 9g of feed were suspended in 1000 mL tap water and 2 mL were added to each 2 L cultures.
- Feeding frequency: daily

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: pipette

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Test temperature:

25 +/- 1 C

pH:

Average >7

Dissolved oxygen:

>69%

Nominal and measured concentrations:

Nominal: 0.4 to 50 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: beakers
- Material, size, fill volume: 400 ml glass beakers filled with 250 mL test water.
- Aeration: none
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control: 4
- Biomass loading rate: 1 animal/50ml

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised water was used to prepare standard artificial medium (synthetic fresh water) as stated by DIN - German Institute of Standardisation
- Culture medium different from test medium: yes, culture medium was de-chlorinated tap water
- Intervals of water quality measurement: 3 times / week

OTHER TEST CONDITIONS
- Photoperiod: 7am to 4pm
- Light intensity: fluorescent lamps - Philis TL 40/25W

EFFECT PARAMETERS MEASURED: mortality, reproduction rate and appearance of offspring, daily.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

ca. 1 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

other: Mortality, reproduction rate, and appearance of offspring

Details on results:

- Mortality of parent animals: 7.1%
- Time to first brood release or time to hatch: 7-8 days for the appearance of first offspring.
- Brood size: 88 offspring

Reported statistics and error estimates:

Student's t-test and U-test for determining the NOEC of reproduction rate and parent mortality.

Primarily, the results were expressed with reference to the nominal concentration. If however, the chemical analysis showed a loss of tested substance greater than 20%, then the lowest analysed concentration (minimum value) obtained during the test was also reported for the NOEC. The 21-d NOEC based on nominal concentrations was 1.6 mg/L. The 21-d NOEC based on the minimum value for 1-Octanol was 1.0 mg/L. The most sensitive endpoint was the appearance of first offspring.

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of the read across study, the 21 d reproduction NOEC value for the constituent, 'linear or branched alcohol' is considered to be 1.0 mg/L.

Executive summary:

A study was conducted to determine the long-term toxicity to aquatic invertebrates of the read across substance, Octan-1-ol (purity not specified), according to the method equivalent or similar to OECD Guideline 211. The read across substance at 0.4 to 50 mg/L nominal concentration was exposed to daphnia magna for 21 days under semi-static conditions in freshwater. Four replicates per read across substance and control group were used, where each vessel contained five test organisms. Samples were taken twice from selected concentration levels of the test series during the test period and analysed chemically. The first sampling was taken before the 7th day previous to any offspring appearance, the second sample was taken between Day 16 and Day 21. No further details on analytical methods were presented. Mortality, reproduction rate and appearance of offspring were evaluated daily. Student's t-test and U-test were used for determining the NOEC of reproduction rate and parent mortality. Primarily, the results were expressed with reference to the nominal concentration. If however, the chemical analysis showed a loss of tested substance greater than 20%, then the lowest analysed concentration (minimum value) obtained during the test was also reported for the NOEC. Based on mortality, reproduction rate and appearance of offspring, the 21-d NOEC was determined to be at 1.6 mg/L (nominal concentration), which was corresponding to 1 mg/L (minimum measured value). The most sensitive endpoint was the appearance of first offspring. Mortality of parent animals were noted to be 7.1%, while time to first brood release or time to hatch was 7-8 days for the appearance of first offspring and brood size was 88 offspring. Therefore, the study validity criteria were fulfilled. Under the study conditions, based on the effects of the read across substance on reproduction and survival of the freshwater test organism Daphnia magna, the 21-day NOEC value was determined to be at 1 mg/L (measured) (Kuhn, 1989). Based on the results of the read across study, similar the 21 d reproduction NOEC values are expected for the constituent, 'linear or branched alcohol'.

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

No data available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

Some exceptions to the OECD method are discussed based on sound technical justifications or empirical observations.

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

All the test concentrations were sampled for chemical analysis three times a week at renewal of the test media (representative samples). An 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 volumes were 200–500 mL. The analyte was extracted from the aqueous test samples by liquid–liquid partitioning with 3–5 mL of n-hexane. After shaking for about 10 min
and settling for 60 min, 100 mL of the supernatant (n-hexane) was added by 50 mL each of the internal standard solution and the derivatizing agent N-methyl-N- trimethylsilyl-trifluoroacetamide (MSTFA puriss for GC, Fluka Chemie GmbH, Buchs, Switzerland). Measurement was performed by GC–MS (Hewlett Packard 5890 Series II plus with MSD 5972 and autosampler 7673) in SIM mode, using internal standard calibration with deuterated C12, C14 or C15 aliphatic alcohol (n-dodecanol-d25, n-tetradecanol-d29, or pentadecanol-d31 in n-hexane by Larodan AB, Sweden) as an internal standard. The calibration solutions of the reference standards were prepared in extra pure n-hexane (Riedel-de Hae¨n AG, Seelze, Germany). The column was a SGE BPX-5 (50 m, ID 0.32 mm, film 0.25 mm). The temperature program consisted of two steps at 60 and 280 +/- 1 deg C maintained for 1 min each when analyzing C14. For C15, 60 and 280 +/- 1 deg C were maintained for 3 and 10 min, respectively. Temperature was adjusted at a rate of 10 +/- 1 deg C/min. The injector temperature was 280 +/- 1 deg C.

Vehicle:

yes

Details on test solutions:

Preparation of the test media
Due to the difficulties in dissolving the test substance and its rapid biodegradation, a method of dosing the test substance had to be used which was not covered by the OECD guideline. The preparation of the test solutions was performed in accordance with guidance given by CONCAWE (1992) and to the ASTM standard D6081-97 (ASTM, 1997) with modifications.

Test media preparation and selection of test concentrations
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. One mixing vessel was used for the preparation of each test concentration. To avoid microbiological degradation of the test substance during the test media preparation, the dilution water was sterilized by heating in an autoclave at 121 +/- 1 deg C for 20 min. The sterilized mixing vessels were then filled with this sterilized water for the preparation of the test solution on a clean bench.


C14 (tetradecanol): The loading of the mixing vessels corresponded to 25, 69, 190, and 500 mg C14 alcohol/L and was targeted to achieve an initial maximum concentration of approximately 350 mg/L. The projected concentration also took into account 30% adsorption to the glassware as shown in pre-studies. This concentration represented the maximum estimate of water solubility (using different models and results of experimental studies, the true value seems to be in the range of 100–350 mg/L). The set-up with 4 concentrations and the spacing factor of 2.7 resulted in the second test concentration corresponding to the most reliable estimate of the water solubility (Fisk et al., 2008) and the lower test concentrations sufficiently below that.

C15 (pentadecanol): The loading of the mixing vessels corresponded to 30, 65, 140 and 300 mg C15 alcohol/L and was targeted to achieve an initial maximum concentration of approximately 200 mg/L. As with C14 alcohol, this accounted for 30% adsorption to the glassware as shown in pre-studies. This concentration represented the maximum estimate of water solubility (using different models and results of experimental studies, the true value is likely to be in the range of
50–200 mg/L). No acute D. magna immobility was observed at initial concentrations up to 500 mg/L (above water solubility according to Fisk et al., 2008) in non-
GLP pre-tests. The set-up with 4 concentrations and the common spacing factor of 2.2 resulted in the second test concentration corresponding to the most reliable estimate of the water solubility (OECD, 2006) and the lowest test concentration sufficiently below that.

For each test concentration, an ethanolic stock solution was prepared. One milliter of the appropriate ethanolic stock solution was pipetted into the 2 L mixing vessel taken from the sterilization process. The still warm mixing vessel was slowly turned to cover a maximum area of the glass walls with the ethanolic 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 star- shaped magnetic stirring bar was placed later.

After the addition of dilution water and 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. Care was taken not to exceed the stirring speed. The control medium was prepared similarly with 1 mL of ethanol. The vessels were kept at room temperature (21 +/-1 deg C). 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 of 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). Rinsing of the test vessels was carried out to saturate the surfaces of the test vessels. After filling, the vessels were closed immediately with 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 h prior to testing.

Test organisms (species):

Daphnia magna

Details on test organisms:

D. magna STRAUS (Crustacea, Phyllopoda, Cladocera) is accepted by OECD (1998a) as a representative freshwater invertebrate. The strain tested originated from the German Federal Environment Agency (clone V) and was bred for many generations in the laboratory of the test facility (Fraunhofer IME, Schmallenberg, Germany). Adult D. magna, at least 3 weeks old, were separated from the stock population by sieving Batches of 30–50 of these adult daphnids were held at approximately 20 +/- 1 deg C in 1.8 L of purified tap water and fed daily with an algal suspension (Desmodesmus subspicatus) and LiquizellR (HOBBY). The water was changed once per week. Newborn D. magna were separated from the adults on a daily basis by sieving. The first brood was not used for testing.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Test temperature:

20 - 22 deg C

pH:

mean pH 9.2 - 9.5

Dissolved oxygen:

>= 4 mg/L

Nominal and measured concentrations:

C14 (tetradecanol): The loading of the mixing vessels corresponded to 25, 69, 190, and 500 ug C14 alcohol/L and was targeted to achieve an initial maximum concentration of approximately 350 ug/L. The projected concentration also took into account 30% adsorption to the glassware as shown in pre-studies. This concentration represented the maximum estimate of water solubility (using different models and results of experimental studies, the true value seems to be in the range of 100–350 mg/L). The set-up with 4 concentrations and the spacing factor of 2.7 resulted in the second test concentration corresponding to the most reliable estimate of the water solubility (Fisk et al., 2008) and the lower test concentrations sufficiently below that.

C15 (pentadecanol): The loading of the mixing vessels corresponded to 30, 65, 140 and 300 ug C15 alcohol/L and was targeted to achieve an initial maximum concentration of approximately 200 ug/L. As with C14 alcohol, this accounted for 30% adsorption to the glassware as shown in pre-studies. This concentration represented the maximum estimate of water solubility (using different models and results of experimental studies, the true value is likely to be in the range of 50–200 ug/L). No acute D. magna immobility was observed at initial concentrations up to 500 ug/L (above water solubility according to Fisk et al., 2008) in non-GLP pre-tests. The set-up with 4 concentrations and the common spacing factor of 2.2 resulted in the second test concentration corresponding to the most reliable estimate of the water solubility (OECD, 2006) and the lowest test concentration sufficiently below that.

Details on test conditions:

Tests were performed in accordance to OECD Test Guideline 211, D. magna Reproduction Test (OECD, 1998a) and under full Good Laboratory Practices. Some exceptions to the OECD method are discussed below based on sound technical justifications or empirical observations. D. magna, 24 h old, were exposed to four concentrations of each test substance and dilution water as control for a period of 21–23 days. The entire test was conducted under sterile conditions as much as possible (e.g., test solutions were added to sterilized test vessels to minimize presence of bacteria and algae that naturally degrade alcohol). The D. magna were introduced and the vessels closed off immediately using an autoclaved silicone stopper. The test solutions were renewed daily by transferring the D. magna to new test vessels containing freshly prepared test media of the same nominal test concentrations. At each transfer, the D. magna were carefully rinsed with sterilized dilution water before being placed in the new vessel. Ten animals were used for each test concentration and for the control. Each D. magna was exposed separately in a numbered vessel containing 100 mL of test medium. The D. magna were fed at each renewal with suspensions of the unicellular green algae, D. subspicatus. Algal suspensions were prepared daily from axenic cultures and analysed for microbial contamination 1 and 2 weeks after test start by using '‘Cult-Dip combis Dip Slides’’ (Merck, Darmstadt, Germany). No bacterial contamination was detected. 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.
The pH value, oxygen concentration, and temperature were checked directly before adding the test organisms, and before and after each renewal. The vessels were subjected to a light/dark cycle of 16/8 h. The temperature during the test was 21 +/- 1 deg C. The light intensity did not exceed 1000 lx. The test vessels were aerated with sterile filtrated synthetic air: the autoclaved silicone stoppers were fitted with fine glass capillaries connected to the aeration unit. At each renewal, the glass capillaries were sterilized by washing with 70% ethanol.
Although in contravention of the OECD test guideline 211, the aeration was necessary to avoid oxygen depletion arising from microbial breakdown of the test substance as demonstrated in pre-studies conducted by Schafers (2005a, b). In the pre-studies it was also shown that an increase in transfer of bacteria was found to be associated with growth of the test organisms during the test. There are two reasons why the OECD recommends that aeration is not used: to avoid evaporation of the test substance and to minimize water movements that could stress the planktonic filter feeders. Test substance evaporation was not a concern because the volatility of the substance is low (Fisk et al., 2008) and the test was run in closed vessels. To minimize movements of the test solution, the capillaries were introduced just below the surface of the test media. It was subsequently verified by observation that the swimming behaviour of the test organisms was not influenced by the aeration.
The test procedures needed to counteract the test substance properties of low aqueous solubility and rapid biodegradability. Test media was renewed daily using autoclaved dilution water. D. magna were rinsed when transferring them to the new test media. Collectively, when these manipulations were combined with aeration of the closed vessels Daphnia could become stressed resulting in lowered reproduction. In the C15 test the number of offspring produced by the control parental organisms in 21 days was below a mean of 60 (test guideline success criterion) but the parents exhibited no significant mortality and looked otherwise healthy. The variability in the numbers of offspring produced was also below 25%, therefore the test was prolonged to 23 days until a mean of 60 newborns per female was achieved. This ensured that the test had equivalent statistical power for discriminating effects. This option was available in the old OECD 202 (part B) (OECD, 1984) and seemed to be justified in this case because of the particular conditions of the test.

Observations and effect data generation
Parental D. magna were visually assessed for immobility and any other abnormalities in appearance and behaviour every day. At study termination, the length of the exposed adults was measured by digital photography and image analysis and compared with those of the control animals. The newborn D. magna in each beaker were counted, inspected for abnormalities and removed at each daily renewal of the test solutions. The following endpoints observed in the reproduction test were evaluated quantitatively: mortality (immobility) of parental generation, 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), and individual length of adults.

Data evaluation and statistical analysis
The evaluation of the concentration effect relationships and the calculations of effect concentrations were based on mean measured initial concentrations as multiple (n ¼ 21–23) peak concentrations, as well as on geometric means between mean measured initial and aged (24 h) test concentrations. For each endpoint, the NOEC, lowest-observed-effect-concentration (LOEC), and, if possible, the EC50, EC20 and EC10 were determined. NOECs and LOECs were calculated by ANOVA followed by Williams’ test (Williams, 1971, 1972) or an appropriate non-parametric test suggested by the ToxRat program (ToxRat Solutions GmbH, Alsdorf, Germany). 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 ToxRat. Analyses for the purposes of (Q)SAR development involved regressions of ecotoxicological endpoints, such as EC10 or NOEC for reproduction effects, versus log Kow of the various alcohols that were tested. Linear regressions were performed using SAS and the generalized linear model procedure (SAS, 2006).

Reference substance (positive control):

not specified

Key result

Duration:

21 d

Dose descriptor:

EC10

Remarks:

Tetradecanol

Effect conc.:

ca. 39 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: Based on mean measured initial concentrations as multiple (n ¼ 21–23) peak concentrations, as well as on geometric means between mean measured initial and aged (24 h) test concentrations

Key result

Duration:

21 d

Dose descriptor:

EC10

Remarks:

Tetradecanol

Effect conc.:

ca. 6.3 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: Based on mean measured initial concentrations as multiple (n ¼ 21–23) peak concentrations, as well as on geometric means between mean measured initial and aged (24 h) test concentrations

Key result

Duration:

21 d

Dose descriptor:

EC10

Remarks:

Pentadecanol

Effect conc.:

> 63 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: Based on mean measured initial concentrations as multiple (n ¼ 21–23) peak concentrations, as well as on geometric means between mean measured initial and aged (24 h) test concentrations

Key result

Duration:

21 d

Dose descriptor:

EC10

Remarks:

Pentadecanol

Effect conc.:

ca. 12 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: Based on mean measured initial concentrations as multiple (n ¼ 21–23) peak concentrations, as well as on geometric means between mean measured initial and aged (24 h) test concentrations

Results

Test substance concentrations

The mean measured test substance concentrations of the initial preparations, for the aged solutions after 1 day and the geometric means of both were given in Table 1. In  spite of the elaborate modifications to reduce losses of test substance, they still occurred as a result of adsorption and degradation by food algae and degradation by adapted bacteria introduced with transferred D. magna, despite thorough rinsing. The Graph (kindly refer to the attached background material section) summarizes representative exposure trends for the low and high doses in C14 chronic studies. Several aspects were apparent. During the first days of the tests, at the low doses, test substance losses within 24 h were between 85% and >95%, whereas at the high doses they were only 50–88%. Beginning around the 5th to 7th day of exposure, it became increasingly difficult to maintain exposures even at the high dose. Exposures declined to near background levels by 24 h. However, the running average (average concentration determined to each point in time) exposure concentration shows the patterns are stable leading to results that can be interpreted meaningfully, in spite of variability in the daily measured data. Based on solubility QSARs and measured aqueous solubility it is likely that some initial higher treatment levels exceeded the level of solubility. During the course of each day, as concentrations declined due to degradative losses, the limits of solubility were crossed. The predicted water solubility (Fisk et al., 2008) was 130 and 49 ug/L for C14, and C15, respectively. Thus, the higher chain lengths were tested in the region of water solubility, and did exceed solubility on some occasions. This is more likely than indicated because water solubility determinations are made in pure water lacking ions, whereas exposures occurred in waters with calcium and magnesium concentrations of 0.65–0.70 and 0.20–0.25 mmol/L, respectively. The expectation is that the solubility of LCOH in environmental waters is less than measured in pure (ion-free) water and that this is especially relevant for the studies with C14 and C15.

Table 1: Mean measured test substance concentrations (ug/L) during Daphnia magna 21 -d chronic reproduction studies

C14	Nominal concentration
	24		69		190		500
	Mean^a	SD%	Mean^a	SD%	Mean^a	SD%	Mean^a SD%
Fresh	10	49	51	21	140	20	370	31
Old	LOQ^c	–         LOQ^c		–	1.2	130	16	160
Mean^b	1.6	3.6			13		77
C15	Nominal concentration
	30		65		140		300
	Mean^a	SD%	Mean^a	SD%	Mean^a	SD%	Mean^a SD %
Fresh   Old   Mean^b	15   0.3   2.0	31   100	56   1.1   7.8	45   160	100   3.3   19	27   150	240   17   63	28   160

a Arithmetic mean of three weekly measurements, representing 21–23 peak concentrations.

b Geometric mean of fresh and old means.

c LOQ was calculated with 0.5 LOQ.

Effects

The mean values for the different test endpoint parameters for each treatment level are listed in Table 2. The NOEC, EC10, EC20 and EC50 values of the biological endpoints are summarized in Table 3.

Table 2: Daphnia magna survival, growth and reproduction data

C14	Survival (%)	Growth (length)	Age at first brood	Cumulative off spring/female	Intrinsic rate of increase,r
		Mean +/- SD (mm)	Mean +/- SD (days)	Mean +/- SD (#)	Mean +/- SD (1/d)
Control	100	4.570.32	7.870.79	8775.7	0.36870.0319
Conc. 1	100	4.870.31	7.770.67	8477.4	0.36470.0343
Conc. 2	100	4.070.38	8.170.74	8079.4*	0.34770.0328
Conc. 3	100	4.570.27	8.270.79	7478.5*	0.33870.0347*
Conc. 4	70	4.770.40	7.970.57	7078.2*	0.33870.0241*
C15	Survival (%)	Growth (length) Mean +/- SD (mm)	Age at first brood Mean +/- SD (days)	Cumulative offspring/female Mean +/- SD (#)	Intrinsic rate of increase,r Mean +/- SD (1/d)
Control	100	3.870.34	9.070.82	63.474.8	0.27270.010
Conc. 1	100	3.870.28	9.070.82	60.774.9	0.26770.018
Conc. 2	100	3.670.33	9.371.06	59.376.9	0.26270.013
Conc. 3	100	3.870.52	9.271.14	54.877.1*	0.25770.018*
Conc. 4	100	3.970.19	8.970.99	50.876.7*	0.25670.020*

*Significant difference to control according to Williams-test (a¼ 0.05, one-sided smaller).

SD ¼ standard deviation. Number of D. magna per concentration: n¼ 10

 

Table 3: Effect of several long-chain alcohols on Daphnia magna reproduction and survival endpoints

C14	Survival	Offspring	Intrinsic rater
EC50initial	360	n.d.	n.d.
Total	130	n.d.	n.d.
EC20initial	230	270	990
Total	58	23	87
EC10initial	180	70	200
Total		6.3	18
NOEC initial	140	9.8	51
Total	13	1.6	3.6
C15	Survival	Offspring	Intrinsic rater
EC50initial	n.d.	n.d.	n.d.
Total	n.d.	n.d.	n.d.
EC20initial	n.d.	240	n.d.
Total	n.d.	62	n.d.
EC10initial	n.d.	74	1200
Total	n.d.	12	500
NOEC initial	240	56	56
Total	63	7.8	7.8

Note: Statistical evaluation of reproductive endpoints only for concentrations without mortality. Effect concentrations of the test substance (ug/L) based on mean measured concentrations. n.d. ¼ not determined (far above concentration range tested and water solubility). Initial ¼ mean measured concentrations at the start of daily exposure. Total ¼ mean of all geometric means of initial and aged concentrations.

 

Survival and growth

During all tests, no parental mortality occurred up to and including the third treatment level and no clinical symptoms were observed. With C10, at the highest treatment level all animals died before they could reproduce. At the highest treatment level with C14, 30% of the 10 introduced D. magna died, whereas with C15 no parental mortality occurred. Adult body length exhibited no significant dose-related effects.

Reproduction and population growth

The mean age at first brood was between 8.9 and 9.4 days for all test substance concentrations of C15, between 8.1 and 8.4 days for C14. There was no statistically significant difference between treatments in any test. No offspring mortality occurred. The cumulative number of offspring was significantly reduced by 14–15% at the third test concentration (mean measured initial concentrations) of C14 (140 ug/L) and C15 (100 ug/L). At the highest treatment with C14 (370 ug/L) and C15 (240 ug/L) (low or no mortality), the decrease of reproduction per surviving female cumulated to 19–20%. With C14, exposure to 51 ug/L resulted in a significant deviation from the controls (8%). The intrinsic rate of increase was significantly reduced at the highest two concentrations of C14 and C15, but by clearly less than 10%. Thus, a reliable EC10 could not be determined by interpolation. In consequence, the cumulative number of  offspring  was the most sensitive endpoint when testing long-chain aliphatic alcohols.

Table 4 Chronic (21-d) EC₁₀values of long-chain alcohols to Daphnia magna

Mean total concentration      Mean initial concentration                     Survival        Reproduction     Survival      Reproduction
	ug/L	mmol	ug/L	mmol	ug/L	mmol	ug/L	mmol
C14	39	0.18	6.3	0.03	180	0.83	70	0.33
C15	>63	>0.28	12	0.05	>240	>1.01	74	0.32

Validity of the tests

All validity criteria for the performance of the controls were fulfilled: survival (100%) was above 80%, the parental D. magna started to reproduce up to day 9 and the mean number of offspring per female was above 60 despite the stressful test conditions (daily transfer and rinsing; aeration). However, for C15, the quality criterion for controls of 60 newborns per female was reached by day 23 (note, however, that survival and other quality criteria were never impaired). The statistical power of the tests was generally high (B <0.2) due to the low variability of reproduction, the coefficients of variation of cumulative offspring number ranging from 6.5% to14% in all treatments without mortality with C14 and C15.

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of the read across study, the 21 d EC10 for the constituent, 'linear or branched alcohol' is considered to be 39 µg/L (mortality) and 6.3 µg/L (reproduction).

Executive summary:

A study was conducted to determine the long term toxicity to aquatic invertebrates of the read across substances,'tetradecanol (Purity: 99.5%) and pentadecanol (Purity: 99.5%)', according to the OECD Guideline 211, in compliance with GLP. Ten Daphnia magna (24 h old) were exposed to four concentrations of each read across substances: 25, 69, 190, 500 µg C14 alcohol/L and 30, 65, 140 and 300 µg C15 alcohol/L and dilution water as control for a period of 21–23 days. The entire test was conducted under sterile conditions as much as possible (e.g., test solutions were added to sterilized test vessels to minimize presence of bacteria and algae that naturally degrade alcohol). Parental Daphnia magna were visually assessed for immobility and any other abnormalities in appearance and behaviour every day. At study termination, the length of the exposed adults was measured by digital photography and image analysis and compared with those of the control animals. The newborn Daphnia magna in each beaker were counted, inspected for abnormalities and removed at each daily renewal of the test solutions. The following endpoints observed in the reproduction test were evaluated quantitatively: mortality (immobility) of parental generation, 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), and individual length of adults. All the test concentrations were sampled for chemical analysis three times a week at renewal of the test media (representative samples). Measurement was performed by GC–MS method and effect concentrations of the read across substance were based on mean measured concentrations. At the highest treatment level, 30% mortality was observed with C14 alcohol and no mortality occurred with C15 alcohol. Adult body length exhibited no significant dose related effects. The mean age at first brood was between for all test concentrations of C15 alcohol and between 8.1 and 8.4 days for C14 alcohol. No offspring mortality occurred. The cumulative number of offspring was significantly reduced by 14-15% at the third test concentrations (190 µg/L for C14 alcohol and 140 µg/L for C15 alcohol). At the highest test concentrations (500 µg/L for C14 alcohol and 300 µg/L for C15 alcohol), the decrease of reproduction per surviving female cumulated to 19-20%. In consequence, the cumulative number of offspring was considered to be the most sensitive endpoint. All validity criteria for the performance of the controls were fulfilled. However, for C15, the quality criterion for controls of 60 newborns per female was reached by Day 23 (although survival and other quality criteria were not impaired). The statistical power of the tests was generally high (B <0.2) due to the low variability of reproduction, the coefficients of variation of cumulative offspring number ranging from 6.5% to 14% in all treatments without mortality with C14 and C15. Under the study conditions, the 21-d EC10 values for C14 and C15 alcohol were determined to be 39 and >63 µg/L (measured) based on mortality and 6.3 and 12 µg/L (measured) based on effects on reproduction respectively (Schafers, 2009). Based on the results of the read across study, similar effect levels are expected for the constituent, 'linear or branched alcohol'.

Description of key information

Overall, data across several alcohols indicate that, alcohols with a carbon chain length greater than C14 are expected to be non-toxic at the solubility limit (Schäfers et al., 2009). Therefore, given the low solubilities of the constituents hexadecan-1-ol (0.0412 mg/L) and isostearyl alcohol (0.01745 mg/L), the L(E)C50 value for the alcohol constituent can be considered to be >0.0412 mg/L.

Additional information

Constituent: Alcohol - read across studies:

Study 1:A study was conducted to determine the long-term toxicity to aquatic invertebrates of the read across substance, 'Octan-1-ol (purity not specified)', according to the method equivalent or similar to OECD Guideline 211. The read across substance at 0.4 to 50 mg/L nominal concentration was exposed to daphnia magna for 21 days under semi-static conditions in freshwater. Four replicates per read across substance and control group were used, where each vessel contained five test organisms. Samples were taken twice from selected concentration levels of the test series during the test period and analysed chemically. The first sampling was taken before the 7th day previous to any offspring appearance, the second sample was taken between Day 16 and Day 21. No further details on analytical methods were presented. Mortality, reproduction rate and appearance of offspring were evaluated daily. Student's t-test and U-test were used for determining the NOEC of reproduction rate and parent mortality. Primarily, the results were expressed with reference to the nominal concentration. If however, the chemical analysis showed a loss of tested substance greater than 20%, then the lowest analysed concentration (minimum value) obtained during the test was also reported for the NOEC. Based on mortality, reproduction rate and appearance of offspring, the 21-d NOEC was determined to be at 1.6 mg/L (nominal concentration), which was corresponding to 1 mg/L (minimum measured value). The most sensitive endpoint was the appearance of first offspring. Mortality of parent animals were noted to be 7.1%, while time to first brood release or time to hatch was 7-8 days for the appearance of first offspring and brood size was 88 offspring. Therefore, the study validity criteria were fulfilled. Under the study conditions, based on the effects of the read across substance on reproduction and survival of the freshwater test organism Daphnia magna, the 21-day NOEC value was determined to be at 1 mg/L (measured) (Kuhn, 1989).

Study 2: A study was conducted to determine the long term toxicity to aquatic invertebrates of the read across substances,'tetradecanol (Purity: 99.5%) and pentadecanol (Purity: 99.5%)', according to the OECD Guideline 211, in compliance with GLP. Ten Daphnia magna (24 h old) were exposed to four concentrations of each read across substances: 25, 69, 190, 500 µg C14 alcohol/L and 30, 65, 140 and 300 µg C15 alcohol/L and dilution water as control for a period of 21–23 days. The entire test was conducted under sterile conditions as much as possible (e.g., test solutions were added to sterilized test vessels to minimize presence of bacteria and algae that naturally degrade alcohol). Parental Daphnia magna were visually assessed for immobility and any other abnormalities in appearance and behaviour every day. At study termination, the length of the exposed adults was measured by digital photography and image analysis and compared with those of the control animals. The newborn Daphnia magna in each beaker were counted, inspected for abnormalities and removed at each daily renewal of the test solutions. The following endpoints observed in the reproduction test were evaluated quantitatively: mortality (immobility) of parental generation, 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), and individual length of adults. All the test concentrations were sampled for chemical analysis three times a week at renewal of the test media (representative samples). Measurement was performed by GC–MS method and effect concentrations of the read across substance were based on mean measured concentrations. At the highest treatment level, 30% mortality was observed with C14 alcohol and no mortality occurred with C15 alcohol. Adult body length exhibited no significant dose related effects. The mean age at first brood was between for all test concentrations of C15 alcohol and between 8.1 and 8.4 days for C14 alcohol. No offspring mortality occurred. The cumulative number of offspring was significantly reduced by 14-15% at the third test concentrations (190 µg/L for C14 alcohol and 140 µg/L for C15 alcohol). At the highest test concentrations (500 µg/L for C14 alcohol and 300 µg/L for C15 alcohol), the decrease of reproduction per surviving female cumulated to 19-20%. In consequence, the cumulative number of offspring was considered to be the most sensitive endpoint. All validity criteria for the performance of the controls were fulfilled. However, for C15, the quality criterion for controls of 60 newborns per female was reached by Day 23 (although survival and other quality criteria were not impaired). The statistical power of the tests was generally high (B <0.2) due to the low variability of reproduction, the coefficients of variation of cumulative offspring number ranging from 6.5% to 14% in all treatments without mortality with C14 and C15. Under the study conditions, the 21-d EC10 values for C14 and C15 alcohol were determined to be 39 and >63 µg/L (measured) based on mortality and 6.3 and 12 µg/L (measured) based on effects on reproduction respectively (Schafers, 2009).

Supporting studies on alcohol from OECD SIDS dossier:

Study 1:A study was conducted to determine the acute toxicity of read across substance, 'Alcohols, C12-18 (purity not specified)', to Daphnia magna, according to the EU Guideline 92/69/EWG, in compliance with GLP. Twenty test organisms were exposed to each nominal test substance concentrations of 0, 10, 30, 100, 1000, 3000 mg/L water accommodated fractions (WAF) for 48 h under static conditions. No solvent was used and instead, the water accommodated fraction (WAF) was used in the test chambers. The concentrations of test substance in the test vessels were measured using the fluid chromatography of dichloromethane extract. Measured concentrations in samples collected from the 10 and 100 mg/L chambers were less than 1% of nominal. The solubility of the lowest carbon chain length in this compound, C12, was 3 mg/L, therefore the EL50 was greater than the solubility limit. Immobilisation was recorded at 24 and 48 h and compared with control values. Based on the study results, the 48 h EL50 was calculated to be 40 mg/L WAF (nominal). As no immobility or other signs of stress were observed in the control and dissolved oxygen concentration remained at 99.3% mg/L, the test met all validity criteria. Under the study conditions, the 48 h EL50 was determined to be 40 mg/L (WAF; nominal) (OECD SIDS, 2006).

Study 2:A study was conducted to determine the acute toxicity of read across substance, '1-Decanol (purity not specified)', to Daphnia magna, according to a method similar to OECD Guideline 202. Twenty daphnia magna were exposed to the range of test substance concentrations for 48 h, under static conditions. No analytical monitoring of the test substance was performed in the study. Immobilisation is recorded at 24 h and 48 h and compared with control values. The results were analysed in order to calculate the EC50 at 48 h. Under the study conditions, the 48 h EC50 and EC100 was determined to be 2.9 and 29 mg/L respectively (nominal) (OECD SIDS, 2006).

Overall, data across several alcohols indicate that, alcohols with a carbon chain length greater than C14 are expected to be non-toxic at the solubility limit (Schäfers et al., 2009). Therefore, given the low solubilities of the constituents hexadecan-1-ol (0.0412 mg/L) and isostearyl alcohol (0.01745 mg/L), the L(E)C50 value for the alcohol constituent can be considered to be >0.0412 mg/L.