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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-01-10 to 2005-01-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Good study conducted following OECD guideline 201 and conducted according to GLP (certified).
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: all nominal concentrations were tested at time 0, 24 and 72 h.

- Sampling method: samples were taken from the vessels with algae and vessels prepared without algae.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: A stock solution was prepared by adding 180 mg of the test substance to sterilised test medium and adjusting the volume to 1 L. The solution was stirred for 24 hours. The stock solution was diluted with sterilised growth medium to obtain the nominal test concentrations under sterile conditions: 7.7, 17, 37, 82 and 180 mg hexanol/L.

- Controls: standard control using dilution water.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM

- Common name: freshwater green alga

- Strain: Pseudokirchneriella subcapitata

- Source (laboratory, culture collection): SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Albrecht von Haller Institut, Untere Klarspüle 2, D-37073 Göttingen, Catalog No 61.81.


ACCLIMATION

- Culturing media and conditions: The stock cultures were maintained fulfilling the criteria of the OECD guidelines. Prior to testing a pre-culture was established in test medium to obtain exponentially growing algae for the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
N.R.
Test temperature:
22.0 - 22.2 °C
pH:
8.52 - 8.58 (Controls), 8.08 - 8.40 (test concentrations)
Dissolved oxygen:
N.R.
Salinity:
N.A.
Nominal and measured concentrations:
Nominal: 7.7, 17, 37, 82 and 180 mg hexanol/L
Mean measured (with algae): 4.72, 11.3, 31.2, 50.1 and 111.2 mg hexanol/L
Mean measured (without algae present): 5.48, 12.3, 29.3, 54.9 and 105.5 mg hexanol/L 
Details on test conditions:
TEST SYSTEM

- Test vessel: glass flask, with cultures being resuspended continuously by shaking on a laboratory shaker (Incubation Shaker Multitron ®, INFORS, Switzerland).

- Type: closed

- Material, size, headspace, fill volume: Test vessels are 250 mL conical glass flasks covered with silicone-sponge caps. The vessels and caps were  sterilized prior to use (autoclaving or heating). 

- Aeration: none

- Initial cells density: 10,000 cells/mL

- Control end cells density: 143.3x10^4 cells/mL

- No. of vessels per concentration: 3

- No. of vessels per control: 6


GROWTH MEDIUM

- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS

- Source/preparation of dilution water: A sterilized synthetic growth medium according to OECD 201

- Culture medium different from test medium: no

- Intervals of water quality measurement: daily


OTHER TEST CONDITIONS

- Sterile test conditions: yes

-Adjustment of pH: none

- Photoperiod: continuous

- Light intensity and quality: 8000 Lux



EFFECT PARAMETERS MEASURED:

- Determination of cell concentrations: Cell concentrations were determined using an electronic particle counter (CASY 1 Model TT Schärfe System
Reutlingen, Germany) from time 0h to 72h every 24h. The correctness of the electronic counts was checked by microscopically counting following internal operation procedures. 


TEST CONCENTRATIONS

- Spacing factor for test concentrations: five concentrations arranged in a geometric series, spaced by a factor of 2.2

- Range finding study: yes, details not reported.
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
11.3 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
31.2 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
19.8 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
79.7 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 4.72 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
< 4.72 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
4.97 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
20.5 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control: yes
Reported statistics and error estimates:
Calculation of the percent inhibition of growth rate, yield (cell counts) and biomass integral was performed according to the OECD guideline.
A concentration-effect relationship was observed and statistically analyzed to obtain effect concentrations.
The test results were statistically analyzed to determine EC10 and EC50 values together with 95 % confidence intervals using Probit-analysis assuming lognormal distribution of the values by using the computer program ToxRat.
The NOEC and LOEC values for yield and biomass were determined by the Welch t-test.

Table 1. Cell densities, biomass and growth rate, and percentage inhibition of the test organism treated at a range of concentrations of the test substance. Biomass and growth rate are the mean at 72 h, the percentage inhibition are expressed relative to the period 0 -72 h.

      Treatment concentration (measured concentration average mg/L)  Cell density              Biomass  Biomass % inhibition  Growth rate Growth rate % inhibition 
 Control 0  Replicate #  24 h 48 h   72 h -  -  -
   1  5.0  27.0  136.3   -  -  -  -
   2  4.5  27.0  138.6   - -  -  -
   3  4.6  29.3  148.6  -  -  -  -
   4  4.9  30.2  148.1  -  -  -  -
   5  4.6  29.7  140.4  -  -  -  -
   6  5.1  27.8  147.7  -  -  -  -
 Mean (± SD)  -  4.8 ± 0.2  28.5 ± 1.4 143.3 ± 5.5   102.4 ±3.8  0  1.655 ± 0.013  0
 4.72 mg/L  1  3.7  26.2  154.5  -  -  -  -
   2  3.1  21.6  137.8  -  -  -  -
   3  1.4  12.1  72.4  -  -  -  -
 Mean (± SD)    2.7 ± 1.2  20.0 ± 7.2  121.6 ± 43.4  81.0 ± 30.0  20.9  1.583 ± 0.136 4.3 
 11.3 mg/L  1  3.0  23.8  145.7  -  -  -  -
   2  3.0  17.5  118.5  -  -  -  -
   3  2.6  16.6  117.0  -  -  -  -
 Mean (± SD)    2.9 ± 0.2  19.3 ± 3.9  127.1 ± 16.2  83.2 ± 12.1 18.7  1.613 ± 0.041  2.5
 31.2 mg/L  1  1.6  2.2  78.5  -  -  -  -
   2  1.7  6.5  43.5  -  -  -  -
   3  2.0  8.8  27.5  -  -  -  -
 Mean ± SD    1.7 ± 0.2  5.8 ± 3.4  49.8 ± 26.1  30.0 ± 9.5  70.7  1.272 ± 0.175  23.1
 50.1 mg/L  1  1.8  7.4 32.6   -  -  -  -
   2  2.0  7.7  36.0  -  -  -  -
   3  1.9  7.1  32.6  -  -  -  -
 Mean (± SD)  -  1.9 ± 0.1  7.4 ± 0.3  33.8 ± 2.0 23.6 ± 1.4  76.9  1.173 ± 0.019  29.1
 111.2 mg/L  1  1.6  4.0  7.8  -  -  -  -
   2  1.7  2.4  4.8  -  -  -  -
   3  1.5  3.5  6.0  -  -  -  -
Mean (± SD)  -  1.6 ± 0.1  3.3 ± 0.8  6.2 ± 1.5  5.5 ± 1.5 94.6  0.063 ± 0.079  63.6

Growth rate was significantly reduced between 50.1 and 111.2 mg/L. The calcuated ErL50 was 79.7 mg/L.

Table 2. Results of analytical monitoring (mg/L).

 Nominal conc. (mg/L)  Sample Replicate #  0h (initial) 24 h  24 h % of initial  72 h  72 h % of initial  Avergae of all measurements
 7.7  with algae  1  4.38  5.4  123  3.7  85  
     2    5.5  126  3.7  85  
     3    6.0  136  5.0  115  
     Mean    5.6  128  4.1  95  4.72
   without algae  4  5.34  5.5  102  5.2  97  
     5    6.00  111  5.7  106  
   mean    5.7  107  5.4  102  5.48
 17  with algae*  1  13.6  11.9  88  7.9  58  
     2    12.9  95  8.7  64  
    3    13.6  100  7.53  56  
     mean    12.8  94  7.53  56  11.3
   without algae  4  13.6 13.3   98  8.0  59  
   5    16.8  124  8.6  64  
     mean    15.0  120  24.1  61  12.3
 37  with algae** 1  32.5  39.0  120  24.1  74  
     2    24.4  75  30.1  94  
   3    40.4  124  26.5  82  
   mean   34.6  107  26.5  81.6  31.2
   without algae  4  32.5  26.3  81  25.7  79.2  
     5    39.4  121  19.4  60  
     mean    32.8  101  22.5  69  29.3
 82 with algae  1  54.7  51.0  92  28.7  26  
   2   62.2  113  31.5  58  
     3    71.1  130  34.2  63  
   mean    61.3  112  34.2  63  50.1
   without algae  4  52.5  78.8  150  42.9  82  
     5    64.6  123  38.4  73  
   mean    71.7  137  40.7  77  54.9
 180  with algae  1  112.9  120.9  107  104.3  92  
     2    86.5  77  113.6  101  
     3    84.9  75  152.2  135  
     mean    97.4  86  123.4  109  111.2
   without algae  4  120.0  70.6  59  121.9  102  
     5    75.6  63  124.7  104  
     mean    73.1  61  123.3  103  105.5

* 0 h measured: 32.0 mg/L; regarded as outlier, omitted from evaluation.

** 0 h measured : 13.5 mg/L; regarded as outlier, omitted from evaluation.

Above are results if the analytical monitoring of the test substance (1 -hexanol) over the experimental duration in all test concentrations with and without algae. The mean measured test concentrations varied in some cases by more than +/-20% of the nominal values. The results are therefore interpreted with respect to the mean measured values. The values in bold are the values effectively used. All values at time 0 h were taken from the test solution prior to the addition of algae. Samples at time 48h and 72h were taken from the exposure vessels.

Validity criteria fulfilled:
yes
Conclusions:
A 72h ErC50 value of 79.7 mg/L and NOEC value of 11.3 mg/L have been determined for the effect of the test substance on growth rate of Pseudokirchnerella subcapitata.

Description of key information

(72 h) ErC50 79.7 mg/L, NOEC 11.3 mg/L, ErC10 19.8 mg/l freshwater algae P. subcapitata.

Key value for chemical safety assessment

EC50 for freshwater algae:
79.7 mg/L
EC10 or NOEC for freshwater algae:
19.8 mg/L

Additional information

A 72h ErC50 of 79.7 mg/L, NOEC of 11.3 mg/L and ErC10 of 19.8 mg/L have been determined for the effect of hexan-1-ol on growth rate of Pseudokirchneriella subcapitata. This study has been chosen as key as it reflects the lowest reliable value available for this endpoint.

A study (Bringmann and Kuhn, 1977) has determined a Toxicity Threshold value of 30 mg/L.

Other studies which have been assigned reliability 4 have determined LOECs in the range 18 to 75 mg/L.

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

The data for single carbon chain length LCAAs show toxicity to increase from an ErC50 of 80 mg/L for C6 to 0.33 mg/L for C12. The C14 and C16 LCAAs were not toxic to algae, indicating a toxicity cut-off at C14 and above. Two of the studies determined NOECs that are suitable for assessing long-term toxicity. A NOEC of 11 mg/L has been reported for hexan-1-ol based on measured exposure concentrations. A test with dodecanol resulted in a 72-h EC0 (equivalent to a NOEC) value of 0.085 mg/L.

Other algal studies used a WAF methodology and/or used test concentrations greater than the limit of water solubility and/or did not report a NOEC or equivalent statistic.

The data for mixed carbon chain length LCAAs (commercial products) shows that substances containing LCAAs with carbon numbers in the ranges of C7-9 to C12-15 exerted toxicity at concentrations of between 3.1 and 0.043-0.092 mg/L. At these concentrations all the constituents are likely to have been fully dissolved. For the C12-18 and C14-15 multi-constituent substances there was evidence of toxic effects in tests conducted on water-accommodated fractions prepared at loading rates that will have exceeded the solubility of some constituents.