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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 - 19 Dec 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP- guideline study
Qualifier:
according to
Guideline:
ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)
Qualifier:
according to
Guideline:
other: ISO 5667-16 (1998) Water Quality Sampling – Guidance on biotesting of samples
Qualifier:
according to
Guideline:
other: US EPA (1985) Methods for measuring the acute toxicity of effluents to freshwater and marine organisms. EPA/600/4-85/013.
GLP compliance:
yes
Remarks:
GLP administered by the UK Dept of Health as stated by the study director.
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: floating and settled particles, were observed after 20 h stirring of 1000 mg/L test item in sea water. Therefore the test item was classified as poorly soluble in sea water and exposures were carried out with water accommodated fractions (WAFs) of the test material. The stocks were prepared and stirred in glass tanks with an appropriate volume of treated seawater and the required weight of test material added. The required volume is then siphoned into the appropriate test tank.
Test organisms (species):
Skeletonema costatum
Details on test organisms:
TEST ORGANISM
- Culture number: C5B2A
- Method of cultivation: pre-cultures in the exponential growth phase were prepared from stock laboratory cultures by inoculating treated seawater with nutrient medium (culture medium) to a cell density of approximately 2 x 10^3 to 10^4 cells/mL. The pre-cultures were incubated at approximately 20 ± 2 °C under constant illumination for 3 d ± 1 d, and were used as the inoculum source for subsequent toxicity tests.

Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
19.2 - 21.6°C
pH:
8.18 - 9.13 (control)
8.05 - 8.16 (test medium initial, after adjustment), 8.22 - 9.16 (test medium after 72 h)
Salinity:
36‰
Nominal and measured concentrations:
Nominal test concentrations: control, 10, 18, 32, 56 and 100 mg/L (loading rate)
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL borosilicate glass conical flasks (all flasks were loosely covered with aluminium foil caps)
- Fill volume: 80 mL of test medium seawater
- Estimated Initial cell density: 9896 cells/mL (initial inoculum was checked microscopically using a haemocytometer)
- Control growth rate: 1.47/d
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: medium is prepared from natural seawater supplied by pump from Scapa Flow, Orkney. All seawater was
UV sterilised and filtered to 0.2 μm. The filtered treated seawater was then enriched with nutrients and vitamins in accordance with ISO guidelines.
- Intervals of water quality measurement: pH was recorded daily

OTHER TEST CONDITIONS
- Adjustment of pH: yes, with 1 M HCl, or NaOH to give a pH of 8±0.2. A stirring period ensured if the pH remained constant.
- Light intensity and quality: 7190 – 8540 Lux. 4 x 40 W tubes were mounted at a distance of approx. 40 cm directly above the test area
- Salinity (for marine algae): 36‰ ± 4‰
- Other: shaker (approx. 18 mm orbit, 150 rpm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counts of algal cell numbers were carried out daily by microscope using either haemocytometer or Sedgewick-Rafter chamber, depending upon cell numbers present or by Fluorometer measurements. Three readings were performed on each test vessel.
- Other: light intensity values were measured daily during the test

TEST CONCENTRATIONS
- Range finding study: yes, 72 h
- Test concentrations: control, 1, 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: based on EL50 (72 h) = 52.81 mg/L (WAF). Concentration range from 10 to 100 mg/L were chosen for the main test.
Reference substance (positive control):
yes
Remarks:
3,5 Dichlorophenol (3.2, 1.8 and 1.0 mg/L)
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
37.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 32.07 - 42.03 mg/L
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
growth rate
Results with reference substance (positive control):
- EC50 (72 h): 2.48 mg/L (95% CI: 2.43 - 2.51 mg/L)
Reported statistics and error estimates:
The results were calculated using ToxCalc Version 5 software.

Table 1: Growth rates after 24, 48 and 72 h

24 h 48 h 72 h
Treatment Replicate Replicate Growth Mean Replicate Growth Mean Replicate Growth Mean
10 a 1 0.99 1.01 1.02 1 0.97
b 0.99 1.02 0.94
18 a 0.73 0.72 0.91 0.91 0.89 0.9
b 0.72 0.92 0.9
32 a 0.32 0.3 0.58 0.55 0.69 0.65
b 0.28 0.53 0.61
56 a 0 0 0 0 0 0
b 0 0 0
100 a 0 0 0 0 0 0
b 0 0 0

Table 2: Effect concentrations in mg/L (nominal, WAF)

95% Confidence limits (mg/L)
EC50 (mg/L) Lower  Upper 72h EC90 (mg/L) 72h NOEC (mg/L)
24 h  25.31 19.12 30.11 - -
48 h 34.18 23.59 41.23 - -
72 h 37.5 32.07 42.03 52.3 10
Endpoint:
toxicity to aquatic algae and cyanobacteria
Adequacy of study:
supporting study
Study period:
12 Jun - 16 Jul 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to
Guideline:
other: European standard NF EN 1484 (July 1997, French version) Water analysis - Guidelines for the determination of total organic carbon (TOC) and dissolved organic carbon (DOC)
GLP compliance:
yes
Remarks:
based on statement of the study director
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: test solutions were prepared from 1 g/L stock solution of the test item previously diluted in algae dilution water
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: CCAP 278.4
- Age of inoculum (at test initiation): 3 d
- Method of cultivation: algae cultures are held in the laboratory on a solid medium (LC/agar petri plates) and in a liquid medium with weekly sub-culturing

ACCLIMATION
- Acclimation period: 3 d prior to the test a preculture of algae was set up in the same conditions as the test, in order to obtain a log phase algae culture for the inoculation. Preculture was checked under a microscope with a heamocytometer.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23 - 23.6 °C
pH:
7.8 - 9.3
Nominal and measured concentrations:
Nominal test concentrations: control, 1, 10 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL erlenmeyer flasks
- Fill volume: 100 mL
- Initial cells density: 10^4 cells/mL
- Control end cells density: 230.5 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (with algae): 6
- No. of vessels per control (without algae): 6

TEST MEDIUM / WATER PARAMETERS
- Ca/Mg ratio: CaCl2 x 2H2O = 18 mg/L / MgSO4 x 7H2O = 15 mg/L, MgCl2 = 12 mg/L
- Intervals of water quality measurement: pH of each concentration were measured at the start and at the end of the test

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- Light intensity: 7682 Lux
- Other: shaking at 120 rpm

EFFECT PARAMETERS MEASURED (with observation intervals if applicable)
- Determination of cell concentrations: algae concentrations were measured every 24 h with a particle counter
Reference substance (positive control):
yes
Remarks:
Potassium Dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Results with reference substance (positive control):
- ErC50 (72 h) = 0.72 mg/L (0.63 - 0.80 mg/L)

Table 1: Results of the TOC analysis of the definitive test in mg/L at the beginning and at the end of the test.

Nominal test item concentrations in mg/L Total Organic Carbon
0 h 72 h
Dilution water control (6 samples) < Algae dilution water Limit Of Quantification (4 mg/L) (0.6/0.6/0.6/0.6/0.6/0.6) < Algae dilution water Limit Of Quantification (4 mg/L) (2.3/2.4/2.5/2.4/6.0*/2.3)
Dilution water control (without Algae, 6 samples) < Algae dilution water Limit Of Quantification (4 mg/L) (0.1/0.0/0.1/0.0/0.0/0.1) < Algae dilution water Limit Of Quantification (2 mg/L) (1.4/1.4/1.4/1.1/1.7/1.4)
100 (3 samples)  mean: 14.4 (14.2/14.6/14.4)  mean: 17.9 (17.4 /17.2/19.1)
* Outlier

The total organic carbon concentrations measured in the test solutions were lower than the algae dilution water Limit of Quantification for the two controls, at the start and the end of the test. The total organic carbon concentration measured in the 100 mg/L concentration has not been satisfactorily maintained within +/- 20% of the nominal concentration during the test.

Table 2: TOC results obtained by the additional non-GLP study. NPOC and TC-IC method are compared.

Total carbon (mg/L) Inorganic carbon (mg/L) Total Organic Carbon (mg/L) NPOC(mg/L)
Replicate mean  Replicate mean  Replicate mean
T0 a 36.4 36.3 a 8.4 8.4 27.9 a 13.3 12.3
b  36.6 b  8.4 b  11.8
c  36 c  8.5 c  11.7
T24 h  a  36.4 36.9 a  9 9 27.9 a  18 17.3
b  37 b 9 b  16.4
c 37.2 c  9 c  17.6
Difference between T0 and T24 h 0% +40%

Results from the additional non-GLP study analyzed TOC by the NPOC and the TC-IC methods. Results from the NPOC method showed increase of the test item concentrations between T0 and T24. It is therefore assumed that the test item is instable in acid medium. TOC measurements performed without sample acidification (TC-IC method) demonstrate that the test item is stable in the aqueous medium.

Table 3: Average algae concentration.

Nominal test item concentration Algae x 10^4/mL at 24 h Algae x 10^4/mL at 48 h Algae x 10^4/mL at 72 h
Average Average Average
Dilution water control 6.7 35.9 230.5
Dilution water control (without algae) 0 0 0
1 mg/L 6.9 38.5 226
10 mg/L 6.6 33.3 176
100 mg/L 4.2 19.4 108

Table 4: Inhibition of the average specific growth rate for each treatment group.

Test item concentration in mg/L 1 10 100
Inhibition of specific growth rate in % replicate a 1.1 4.2 12.7
replicate b -0.6 5.5 14.1
replicate c 0.7 5.2 15
Inhibition of average specific growth rate in % 0.4 5 13.9

A slight inhibition of the average growth rate was observed. The maximum inhibition of the specific growth rate is 15%.

Validity criteria:

  • pH change < 1.5 in controls
  • Biomass in the control cutlures should have increased by a factor of at least 16 within the 72 h test period – in this study 230.5%
  • Coefficient of variation of the average specific growth rate during the whole test period in replicates control cutlures must not exceed 7% - in this study 1%
  • Coefficient variation of the daily average growth rate of controls must not exceed 35% - in this study 8.6%
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Insuficient data available (only key results are summarized in the report, no description of the methods, no reference on the inoculum, no reference on analytics is given)
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
not specified
Analytical monitoring:
not specified
Vehicle:
not specified
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23 ± 2 °C
Nominal and measured concentrations:
Nominal test concentrations: control, 350, 700, 1000, 1500 and 2000 mg/L (nominal)
Details on test conditions:
OTHER TEST CONDITIONS
- Photoperiod: permanent illumination
- Light intensity and quality: 8000 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: at 24, 48 and 72 h
Reference substance (positive control):
not specified
Duration:
72 h
Dose descriptor:
IC50
Effect conc.:
1 250 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
IC10
Effect conc.:
560 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate

Talbe 1: Results of the cell division inhibition test

Test item concentration in mg/L 350 700 1000 1500 2000
Inhibition of growth rate [%] 0 20 40 60 75

Description of key information

EL50 (72 h) = 37.5 mg/L (nominal, WAF) for Skeletonema costatum based on growth rate (ISO 10253, ISO 5667-16, EPA/600/4-85/013)
NOELR (72 h) = 10 mg/L (nominal, WAF) for Skeletonema costatum based on growth rate (ISO 10253, ISO 5667-16, EPA/600/4-85/013)

Key value for chemical safety assessment

Additional information

Three studies, in which the toxicity of D-Glucopyranose, oligomeric, undecyl glycoside (CAS 98283-67-1) to algae was investigated, are available.

 

The toxicity of the substance was tested to algae according to ISO 10253, ISO 5667-16, EPA/600/4-85/013 and GLP (Opus Plus Limited, 2009). The marine alga Skeletonema costatum was exposed to the test substance for 72 h, at the nominal test loading rates of 10, 18, 32, 56 and 100 mg/L. The test solutions were prepared as water accommodated fractions (WAF), as the test item was found to be poorly soluble in saltwater. No analytical monitoring was performed in this test. Biological effects were observed and resulted in NOELR and EL50 values of 10 mg/L and 37.5 mg/L (nominal, WAF), respectively, based on growth rate.

 

A further study of reliability 1 supports the above mentioned results. The study was performed according to OECD guideline 201 and GLP (FCBA, 2011). The test organism Pseudokirchneriella subcapitata was exposed to the test substance in a static system for 72 h, at nominal test concentrations of 1, 10 and 100 mg/L. The test item was analytically determined by TOC measurements at the beginning and at the end of the study for the highest test concentration. An EC50 (72 h) value of >100 mg/L was determined. Although the study was valued as reliable 1, it is used only as supporting study since a NOEC has not been derived and the study by Hudson (2009) provides the lowest effect value.

 

The results of a study conducted according to OECD guideline 201 are summarized in a very brief report (Hygiene Institut, 1999), which was assigned a reliability of 4, due to insufficient data. Desmodesmus subspicatus as the test alga, was exposed to nominal test item concentrations of 350, 700, 1000, 1500 and 2000 mg/L for 72 h. No analytical monitoring was performed. The study resulted in an IC50 (72 h) and an IC10 (72 h) of 1250 mg/L and 560 mg/L (nominal), respectively, based on the growth rate of the algae.

 

On the basis of the available results the NOELR showing long term effects on algae was determined to be 10 mg/L (nominal, WAF) while the EL50 showing short term effects to algae was calculated to be 37.5 mg/L (nominal, WAF).