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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
long-term saltwater toxicity test of macroalgae
GLP compliance:
not specified
Specific details on test material used for the study:
Reagent grade sodium arsenate HAsNa2O4·7H2O
source: SigmaChemical(St.LouisMO)
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
Reagent grade sodium arsenate (NazHAsO4 •7H20) was obtained from SigmaChemical (St.LouisMO), and stock solutions were made in distilled water at (0.01 g/ml).
test medium: concentrated (Sx) artificial seawater
Test organisms (species):
other: Macrocystis pyrifera
Details on test organisms:
M. pyrifera sporophylls were collected from kelp beds near Carpinteria, Santa Barbara County or near Oxnard,Ventura County (California,USA) and shipped at 4°C to arrive at the University of California, Bodega Marine Laboratory within 24h. Upon arrival, sporophylls were rinsed with seawater, allowed to air dry, and then immersed in filtered seawater to induce zoospore release.
Zoospores were counted by hemocytometer and dispensed at a density of approximately 10000 spores per ml.

Zoospores were cultured at 15°C in a Revco® incubator, illuminated by continuous 15-W cool fluorescent light at 100micro E m-2 s-1.
Cultures were nmonitored until nuclear migration was completed in controls (approx. 42 h), then fixed with 0.15% glutaraldehyde(final concentration) in seawater.
The culture medium consisted of FSW (filtered seawater) (pH 7.8) with Penicillin-G (0.01%
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
42 h
Test temperature:
15 °C
pH:
7.8
Salinity:
19 parts per thousand
Nominal and measured concentrations:
Confirmation of test concentration: 93% to 106% of nominal As
Exact concentraiton were not reported, only shown on graphs.
Details on test conditions:
As experiments were conducted in polystyrene plastic tissue culture wells.
The final volume in all experimental containers was either 5 or 10 ml. All exposureswere typically static (continuous exposurefor 42 h).
Batches of zoospores were considered to be experimental unit, experiments were replicated three times.
Reference substance (positive control):
no
Key result
Duration:
42 h
Dose descriptor:
NOEC
Effect conc.:
40 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
dissolved
Basis for effect:
other: germination
Key result
Duration:
42 h
Dose descriptor:
NOEC
Effect conc.:
40 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
other: germ tube growth
Validity criteria fulfilled:
not specified
Conclusions:
A NOEC of 40 µg As /L was reported for the marine macroalga Macrocystis pyrifera covering endpoints related to reproduction (germination, germ tube growth).
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
The effect of arsenic on the growth of the marine alga Fucus serratus was assessed in a 13-week toxicity test by measuring the length of the longest frond at weeks 0 and 13.
GLP compliance:
not specified
Specific details on test material used for the study:
Arsenate (as Na2HAsO4)
Analytical monitoring:
yes
Details on sampling:
Samples were taken daily for the first 7 days, and then weekly. Water samples were analyzed without pretreatment.
Vehicle:
no
Details on test solutions:
natural water (Kerteminde flord)
18Arsenate stock solutions were prepared from Na2HAsO4 at concentrations of 30.0, 75.0, and 150.0 µg As/ml.
Test organisms (species):
other: Fucus serratus
Details on test organisms:
TEST ORGANISM
- Source (laboratory, culture collection): Algae were collected from Fyns Hoved, Denmark, in May 1999. Individuals (30–50-cm maximum frond length) attached to small stones were transported in seawater to an aquarium facility at Kerteminde.
- Method of cultivation: Algae were cleaned of epiphytes, and 16 individuals were measured (maximum frond length to within 0.5 cm) and randomly distributed equally among four glass aquaria (40 3 80 3 40 cm high) each containing 100 L of seawater. The algae were kept attached to the stones to minimize damage and to ensure that they occupied the full depth of water and were not merely floating on the surface.

ACCLIMATION
- Acclimation period: not specified
- Culturing media and conditions: same as test
Test type:
flow-through
Water media type:
saltwater
Limit test:
no
Total exposure duration:
19 wk
Test temperature:
16-20 °C
Salinity:
12.5 to 22.0 ‰ with a median value of 15.9 ‰
Nominal and measured concentrations:
Nominal concentrations of 0, 20, 50, 100 µg As/ L correspond to measured concentrations of ca. 1, 18.7, 49.6, 96.6 µg As/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: glass aquaria (40 x 80 x 40 cm high)
- Fill volume: 100 L seawater
- Type of flow-through: peristaltic pump
- Renewal rate of test solution (flow rate): 2.0 ml/h for arsenate solution; 3.0 L/h for seawater
- No. of organisms per vessel: 4 individuals
- No. of vessels per concentration (replicates): no true replicates
- No. of vessels per control (replicates): no true replicates

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Seawater from the Kerteminde fjord (stored in a settling tank (130-L capacity) to remove silt and extraneous material before being distributed to the experimental tanks)

- Photoperiod: 14:10 h light:dark
- Light intensity and quality: artifical light, 300-400 µmol/m²/s
- Salinity (for marine algae): salinity ranged from 12.5 to 22.0‰ with a median value of 15.9‰

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Other: Frond number

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ≤ 2.5
Reference substance (positive control):
no
Key result
Duration:
13 wk
Dose descriptor:
NOEC
Effect conc.:
18.7 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
other: length of the longest frond
Details on results:
The growth of individual F. serratus was recorded by measuring the length of the longest frond at weeks 0 and 13. At week 0, no significant differences were observed in the mean lengths of algae from the four groups. At week 13, the mean increases in length for the four groups were as follows: group 0 (5.3 cm), group 20 (7.3 cm), group 50 (3.0 cm), and group 100 (0 cm). The increases in length for group 20 were significantly higher (p < 0.01) than those recorded for group 50 but were not significantly different from group 0. The growth rates of the group 0 and group 20 algae were: 0.41 cm/week and 0.56 cm/week.
Reported statistics and error estimates:
Statistical differences between groups were tested for significance (α = 0.05) with a t test or analysis of variance.
Validity criteria fulfilled:
not specified
Remarks:
Validity is not assessable
Conclusions:
The 13-wk NOEC value reported for saltwater macro-algae is 18.7 µg As/L for the the growth of Fucus serratus.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
7 days long chronic static toxicity test on Achnanthidium minutissimum
GLP compliance:
no
Specific details on test material used for the study:
Special-grade As2O3 (Wako, Osaka, Japan)
Analytical monitoring:
yes
Details on sampling:
On a pre-specified day, samples were collected and filtered through 0.45-μm membrane filters under low vacuum pressure (<30 mm Hg). The filtrate sample was then stored in a cool and dark place after adding 0.50 mL of 1.0 mol/L HCl to them. After that, 20 mL of 3.0 mol/L HCl was added to the sample, and the mixture was heated on a hot plate at 100 °C for 3 h to extract the arsenic both inside and outside of the freshwater phytoplankton cells. Thereafter, the mixture volume was adjusted to 40 mL with ultrapure water, and it was also adjusted to the same concentration as that of 1.3 mol/L HCl (pH = 0).
Vehicle:
no
Details on test solutions:
CSi medium was used for the maintenance of the studied freshwater phytoplankton strains. For the growth experiment, the phosphate concentrations of the CSi medium were adjusted to 1.0 μmol/L or 50 μmol/L.
Test organisms (species):
other: Achnanthidium minutissimum
Details on test organisms:
Achnanthidium minutissimum was obtained from the National Institute for Environmental Studies, Japan.
initial cell density: <4.6*10^3 cells/mL
Phytoplankton cells acclimated to culture media at the exponential growth phase were incubated in 60-mL capacity polycarbonate vessels containing 60 mL of sterilized CSi culture medium (Table 2). The culture medium was then modified by changing the concentration
of phosphate (added as KH2PO4) to encourage the uptake of arsenic species.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d
Test temperature:
25 °C
pH:
7
Nominal and measured concentrations:
1.5, 7.5, 74.9, 749 and 7492 µg As/L
Details on test conditions:
Phytoplankton cells acclimated to culture media at the exponential growth phase were incubated in 60-mL capacity polycarbonate vessels containing 60 mL of sterilized CSi culture medium. The culture medium was then modified by changing the concentration of phosphate (added as KH2PO4) to encourage the uptake of arsenic species. Two different arsenic concentration treatments, high- arsenic ([As]0 = 1.0 μmol/L) and low-arsenic ([As]0 = 20 nmol/L), were used in the experimen in which As was profided in the form of NaH2AsO4.
Initially, the cell density in the culture medium was less than 4.6×10^3 cells/mL. After incubating the phytoplankton for three days, arsenic was added to the culture medium. The cultures were grown for 30 days. Phytoplankton growth was measured spectrophotometrically using a UV-VIS (ultraviolet-visible) spectrophotometer at 540 nm. The cell number was estimated with an established cell density-to-absorbance ratio. The number of cells was counted directly under a microscope. The growth rate (%) was defined by the following equation:
Growth rate[%]=(ODSample/ODControl)×100
where ODSample is the optical density of phytoplankton at 540 nm in arsenic-containing media and ODControl is the optical density of phytoplankton in arsenic-free media after seven days of cultivation.
Key result
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
749 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Reported statistics and error estimates:
NOEC using Duncan multiple range tests
Validity criteria fulfilled:
not specified
Conclusions:
The 7 days long static toxicity test on Achnanthidium minutissimum has yielded the following results:
NOEC (growht rate): 749 µg As / L
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
7 days long chronic static toxicity test on various phytoplankton species
GLP compliance:
not specified
Specific details on test material used for the study:
Special-grade HAsNa2O4·7H2O (Wako, Osaka, Japan)
Analytical monitoring:
yes
Details on sampling:
On a pre-specified day, samples were collected and filtered through 0.45-μm membrane filters under low vacuum pressure (<30 mm Hg). The filtrate sample was then stored in a cool and dark place after adding 0.50 mL of 1.0 mol/L HCl to them. After that, 20 mL of 3.0 mol/L HCl was added to the sample, and the mixture was heated on a hot plate at 100 °C for 3 h to extract the arsenic both inside and outside of the freshwater phytoplankton cells. Thereafter, the mixture volume was adjusted to 40 mL with ultrapure water, and it was also adjusted to the same concentration as that of 1.3 mol/L HCl (pH = 0).
Vehicle:
no
Details on test solutions:
CSi medium was used for the maintenance of the studied freshwater phytoplankton strains. For the growth experiment, the phosphate concentrations of the CSi medium were adjusted to 1.0 μmol/L or 50 μmol/L.
Test organisms (species):
other: Achnanthidium minutissimum
Details on test organisms:
diatom
Achnanthidium minutissimum was obtained from the National Institute for Environmental Studies, Japan.
initial cell density: <4.6*10^3 cells/mL
Phytoplankton cells acclimated to culture media at the exponential growth phase were incubated in 60-mL capacity polycarbonate vessels containing 60 mL of sterilized CSi culture medium. The culture medium was then modified by changing the concentration of phosphate (added as KH2PO4) to encourage the uptake of arsenic species.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d
Test temperature:
25 °C
pH:
7
Nominal and measured concentrations:
1.5, 7.5, 74.9, 749 and 7492 µg As/L
Details on test conditions:
Phytoplankton cells acclimated to culture media at the exponential growth phase were incubated in 60-mL capacity polycarbonate vessels containing 60 mL of sterilized CSi culture medium. The culture medium was then modified by changing the concentration of phosphate (added as KH2PO4) to encourage the uptake of arsenic species. Two different arsenic concentration treatments, high- arsenic ([As]0 = 1.0 μmol/L) and low-arsenic ([As]0 = 20 nmol/L), were used in the experimen in which As was profided in the form of NaH2AsO4.
Initially, the cell density in the culture medium was less than 4.6×10^3 cells/mL. After incubating the phytoplankton for three days, arsenic was added to the culture medium. The cultures were grown for 30 days. Phytoplankton growth was measured spectrophotometrically using a UV-VIS (ultraviolet-visible) spectrophotometer at 540 nm. The cell number was estimated with an established cell density-to-absorbance ratio. The number of cells was counted directly under a microscope. The growth rate (%) was defined by the following equation:
Growth rate[%]=(ODSample/ODControl)×100
where ODSample is the optical density of phytoplankton at 540 nm in arsenic-containing media and ODControl is the optical density of phytoplankton in arsenic-free media after seven days of cultivation.
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
749 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
122 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
38 669 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Reported statistics and error estimates:
EC10 and EC50 have been re-calculated using Trap software (log-logistic curve fitting)
NOEC using Duncan multiple range tests
Validity criteria fulfilled:
not specified
Conclusions:
The 7 days long static toxicity test on Achnanthidium minutissimum has yielded the following results:
NOEC (growth rate): 749 µg As /L
EC10 (growth rate): 122 µg As /L
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
7 days long chronic static toxicity test on various phytoplankton species
GLP compliance:
no
Specific details on test material used for the study:
Special-grade disodium hydrogen formate heptahydrate (AsV) (Wako, Osaka, Japan)
Analytical monitoring:
yes
Details on sampling:
On a pre-specified day, samples were collected and filtered through 0.45-μm membrane filters under low vacuum pressure (<30 mm Hg). The filtrate sample was then stored in a cool and dark place after adding 0.50 mL of 1.0 mol/L HCl to them. After that, 20 mL of 3.0 mol/L HCl was added to the sample, and the mixture was heated on a hot plate at 100 °C for 3 h to extract the arsenic both inside and outside of the freshwater phytoplankton cells. Thereafter, the mixture volume was adjusted to 40 mL with ultrapure water, and it was also adjusted to the same concentration as that of 1.3 mol/L HCl (pH = 0).
Vehicle:
no
Details on test solutions:
CSi medium was used for the maintenance of the studied freshwater phytoplankton strains. For the growth experiment, the phosphate concentrations of the CSi medium were adjusted to 1.0 μmol/L or 50 μmol/L.
Test organisms (species):
other: Botryococcus braunii
Details on test organisms:
Botryococcus braunii was obtained from the National Institute for Environmental Studies, Japan.
initial cell density: <4.6*10^3 cells/mL
Phytoplankton cells acclimated to culture media at the exponential growth phase were incubated in 60-mL capacity polycarbonate vessels containing 60 mL of sterilized CSi culture medium (Table 2). The culture medium was then modified by changing the concentration
of phosphate (added as KH2PO4) to encourage the uptake of arsenic species.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d
Test temperature:
25 °C
pH:
7
Nominal and measured concentrations:
1.5, 7.5, 74.9, 749 and 7492 µg As/L
Details on test conditions:
Phytoplankton cells acclimated to culture media at the exponential growth phase were incubated in 60-mL capacity polycarbonate vessels containing 60 mL of sterilized CSi culture medium. The culture medium was then modified by changing the concentration of phosphate (added as KH2PO4) to encourage the uptake of arsenic species. Two different arsenic concentration treatments, high- arsenic ([As]0 = 1.0 μmol/L) and low-arsenic ([As]0 = 20 nmol/L), were used in the experimen in which As was profided in the form of NaH2AsO4.
Initially, the cell density in the culture medium was less than 4.6×10^3 cells/mL. After incubating the phytoplankton for three days, arsenic was added to the culture medium. The cultures were grown for 30 days. Phytoplankton growth was measured spectrophotometrically using a UV-VIS (ultraviolet-visible) spectrophotometer at 540 nm. The cell number was estimated with an established cell density-to-absorbance ratio. The number of cells was counted directly under a microscope. The growth rate (%) was defined by the following equation:
Growth rate[%]=(ODSample/ODControl)×100
where ODSample is the optical density of phytoplankton at 540 nm in arsenic-containing media and ODControl is the optical density of phytoplankton in arsenic-free media after seven days of cultivation.
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
749 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Reported statistics and error estimates:
NOEC using Duncan multiple range tests
Validity criteria fulfilled:
not specified
Conclusions:
The 7 days long static toxicity test on Botryococcus braunii has yielded the following results:
NOEC (growht rate): 749 µg As / L
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
7 days long chronic static toxicity test on Closterium aciculare
GLP compliance:
no
Specific details on test material used for the study:
Special-grade As2O3 (Wako, Osaka, Japan)
Analytical monitoring:
yes
Details on sampling:
On a pre-specified day, samples were collected and filtered through 0.45-μm membrane filters under low vacuum pressure (<30 mm Hg). The filtrate sample was then stored in a cool and dark place after adding 0.50 mL of 1.0 mol/L HCl to them. After that, 20 mL of 3.0 mol/L HCl was added to the sample, and the mixture was heated on a hot plate at 100 °C for 3 h to extract the arsenic both inside and outside of the freshwater phytoplankton cells. Thereafter, the mixture volume was adjusted to 40 mL with ultrapure water, and it was also adjusted to the same concentration as that of 1.3 mol/L HCl (pH = 0).
Vehicle:
no
Details on test solutions:
CSi medium was used for the maintenance of the studied freshwater phytoplankton strains. For the growth experiment, the phosphate concentrations of the CSi medium were adjusted to 1.0 μmol/L or 50 μmol/L.
Test organisms (species):
other: Closterium aciculare
Details on test organisms:
Closterium aciculare was isolated from Lake Biwa) was provided by Dr. Kanako Naito of Prefectural University of Hiroshima, Japan.
initial cell density: <4.6*10^3 cells/mL
Phytoplankton cells acclimated to culture media at the exponential growth phase were incubated in 60-mL capacity polycarbonate vessels containing 60 mL of sterilized CSi culture medium (Table 2). The culture medium was then modified by changing the concentration
of phosphate (added as KH2PO4) to encourage the uptake of arsenic species.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d
Test temperature:
25 °C
pH:
7
Nominal and measured concentrations:
1.5, 7.5, 74.9, 749 and 7492 µg As/L
Details on test conditions:
Phytoplankton cells acclimated to culture media at the exponential growth phase were incubated in 60-mL capacity polycarbonate vessels containing 60 mL of sterilized CSi culture medium. The culture medium was then modified by changing the concentration of phosphate (added as KH2PO4) to encourage the uptake of arsenic species. Two different arsenic concentration treatments, high- arsenic ([As]0 = 1.0 μmol/L) and low-arsenic ([As]0 = 20 nmol/L), were used in the experimen in which As was profided in the form of NaH2AsO4.
Initially, the cell density in the culture medium was less than 4.6×10^3 cells/mL. After incubating the phytoplankton for three days, arsenic was added to the culture medium. The cultures were grown for 30 days. Phytoplankton growth was measured spectrophotometrically using a UV-VIS (ultraviolet-visible) spectrophotometer at 540 nm. The cell number was estimated with an established cell density-to-absorbance ratio. The number of cells was counted directly under a microscope. The growth rate (%) was defined by the following equation:
Growth rate[%]=(ODSample/ODControl)×100
where ODSample is the optical density of phytoplankton at 540 nm in arsenic-containing media and ODControl is the optical density of phytoplankton in arsenic-free media after seven days of cultivation.
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
749 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
25.5 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
6 646 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Reported statistics and error estimates:
NOEC using Duncan multiple range tests
EC10 have been re-calculated using Trap software (log-logistic curve fitting)
Validity criteria fulfilled:
not specified
Conclusions:
The 7 days long static toxicity test on Closterium aciculare has yielded the following results:
NOEC (growht rate): 749 µg As/ L
EC10 (growht rate): 25.5 µg As/ L
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
7 days long chronic static toxicity test on various phytoplankton species
GLP compliance:
no
Specific details on test material used for the study:
Special-grade disodium hydrogen formate heptahydrate (AsV) (Wako, Osaka, Japan)
Analytical monitoring:
yes
Details on sampling:
On a pre-specified day, samples were collected and filtered through 0.45-μm membrane filters under low vacuum pressure (<30 mm Hg). The filtrate sample was then stored in a cool and dark place after adding 0.50 mL of 1.0 mol/L HCl to them. After that, 20 mL of 3.0 mol/L HCl was added to the sample, and the mixture was heated on a hot plate at 100 °C for 3 h to extract the arsenic both inside and outside of the freshwater phytoplankton cells. Thereafter, the mixture volume was adjusted to 40 mL with ultrapure water, and it was also adjusted to the same concentration as that of 1.3 mol/L HCl (pH = 0).
Vehicle:
no
Details on test solutions:
CSi medium was used for the maintenance of the studied freshwater phytoplankton strains. For the growth experiment, the phosphate concentrations of the CSi medium were adjusted to 1.0 μmol/L or 50 μmol/L.
Test organisms (species):
other: Closterium aciculare
Details on test organisms:
Closterium aciculare (isolated from Lake Biwa) was provided by Dr. Kanako Naito of Prefectural University of Hiroshima, Japan.
initial cell density: <4.6*10^3 cells/mL
Phytoplankton cells acclimated to culture media at the exponential growth phase were incubated in 60-mL capacity polycarbonate vessels containing 60 mL of sterilized CSi culture medium (Table 2). The culture medium was then modified by changing the concentration
of phosphate (added as KH2PO4) to encourage the uptake of arsenic species.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d
Test temperature:
25 °C
pH:
7
Nominal and measured concentrations:
1.5, 7.5, 74.9, 749 and 7492 µg As/L
Details on test conditions:
Phytoplankton cells acclimated to culture media at the exponential growth phase were incubated in 60-mL capacity polycarbonate vessels containing 60 mL of sterilized CSi culture medium. The culture medium was then modified by changing the concentration of phosphate (added as KH2PO4) to encourage the uptake of arsenic species. Two different arsenic concentration treatments, high- arsenic ([As]0 = 1.0 μmol/L) and low-arsenic ([As]0 = 20 nmol/L), were used in the experimen in which As was profided in the form of NaH2AsO4.
Initially, the cell density in the culture medium was less than 4.6×10^3 cells/mL. After incubating the phytoplankton for three days, arsenic was added to the culture medium. The cultures were grown for 30 days. Phytoplankton growth was measured spectrophotometrically using a UV-VIS (ultraviolet-visible) spectrophotometer at 540 nm. The cell number was estimated with an established cell density-to-absorbance ratio. The number of cells was counted directly under a microscope. The growth rate (%) was defined by the following equation:
Growth rate[%]=(ODSample/ODControl)×100
where ODSample is the optical density of phytoplankton at 540 nm in arsenic-containing media and ODControl is the optical density of phytoplankton in arsenic-free media after seven days of cultivation.
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
7.5 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
4.6 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
30.2 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Reported statistics and error estimates:
EC10 have been re-calculated using Trap software (log-logistic curve fitting)
NOEC using Duncan multiple range tests
Validity criteria fulfilled:
not specified
Conclusions:
The 7 days long static toxicity test on Closterium aciculare has yielded the following results:
NOEC (growht rate): 7.5 µg As / L
EC10 (growth rate): 4.6 µg As / L
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
7 days long chronic static toxicity test on various phytoplankton species
GLP compliance:
no
Specific details on test material used for the study:
Special-grade disodium hydrogen formate heptahydrate (AsV) (Wako, Osaka, Japan)
Analytical monitoring:
yes
Details on sampling:
On a pre-specified day, samples were collected and filtered through 0.45-μm membrane filters under low vacuum pressure (<30 mm Hg). The filtrate sample was then stored in a cool and dark place after adding 0.50 mL of 1.0 mol/L HCl to them. After that, 20 mL of 3.0 mol/L HCl was added to the sample, and the mixture was heated on a hot plate at 100 °C for 3 h to extract the arsenic both inside and outside of the freshwater phytoplankton cells. Thereafter, the mixture volume was adjusted to 40 mL with ultrapure water, and it was also adjusted to the same concentration as that of 1.3 mol/L HCl (pH = 0).
Vehicle:
no
Details on test solutions:
CSi medium was used for the maintenance of the studied freshwater phytoplankton strains. For the growth experiment, the phosphate concentrations of the CSi medium were adjusted to 1.0 μmol/L or 50 μmol/L.
Test organisms (species):
other: Pediastrum duplex
Details on test organisms:
Pediastrum duplex was obtained from the National Institute for Environmental Studies, Japan.
initial cell density: <4.6*10^3 cells/mL
Phytoplankton cells acclimated to culture media at the exponential growth phase were incubated in 60-mL capacity polycarbonate vessels containing 60 mL of sterilized CSi culture medium (Table 2). The culture medium was then modified by changing the concentration
of phosphate (added as KH2PO4) to encourage the uptake of arsenic species.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d
Test temperature:
25 °C
pH:
7
Nominal and measured concentrations:
1.5, 7.5, 74.9, 749 and 7492 µg As/L
Details on test conditions:
Phytoplankton cells acclimated to culture media at the exponential growth phase were incubated in 60-mL capacity polycarbonate vessels containing 60 mL of sterilized CSi culture medium. The culture medium was then modified by changing the concentration of phosphate (added as KH2PO4) to encourage the uptake of arsenic species. Two different arsenic concentration treatments, high- arsenic ([As]0 = 1.0 μmol/L) and low-arsenic ([As]0 = 20 nmol/L), were used in the experimen in which As was profided in the form of NaH2AsO4.
Initially, the cell density in the culture medium was less than 4.6×10^3 cells/mL. After incubating the phytoplankton for three days, arsenic was added to the culture medium. The cultures were grown for 30 days. Phytoplankton growth was measured spectrophotometrically using a UV-VIS (ultraviolet-visible) spectrophotometer at 540 nm. The cell number was estimated with an established cell density-to-absorbance ratio. The number of cells was counted directly under a microscope. The growth rate (%) was defined by the following equation:
Growth rate[%]=(ODSample/ODControl)×100
where ODSample is the optical density of phytoplankton at 540 nm in arsenic-containing media and ODControl is the optical density of phytoplankton in arsenic-free media after seven days of cultivation.
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
7.5 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
4.6 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
72.5 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Reported statistics and error estimates:
EC10 have been re-calculated using Trap software (log-logistic curve fitting)
NOEC using Duncan multiple range tests
Validity criteria fulfilled:
not specified
Conclusions:
The 7 days long static toxicity test on Pediastrum duplex has yielded the following results:
NOEC (growht rate): 7.5 µg As/ L
EC10 (growth rate): 4.6 µg As / L
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
7 days long chronic static toxicity test on various phytoplankton species
GLP compliance:
no
Specific details on test material used for the study:
Special-grade disodium hydrogen formate heptahydrate (AsV) (Wako, Osaka, Japan)
Analytical monitoring:
yes
Details on sampling:
On a pre-specified day, samples were collected and filtered through 0.45-μm membrane filters under low vacuum pressure (<30 mm Hg). The filtrate sample was then stored in a cool and dark place after adding 0.50 mL of 1.0 mol/L HCl to them. After that, 20 mL of 3.0 mol/L HCl was added to the sample, and the mixture was heated on a hot plate at 100 °C for 3 h to extract the arsenic both inside and outside of the freshwater phytoplankton cells. Thereafter, the mixture volume was adjusted to 40 mL with ultrapure water, and it was also adjusted to the same concentration as that of 1.3 mol/L HCl (pH = 0).
Vehicle:
no
Details on test solutions:
CSi medium was used for the maintenance of the studied freshwater phytoplankton strains. For the growth experiment, the phosphate concentrations of the CSi medium were adjusted to 1.0 μmol/L or 50 μmol/L.
Test organisms (species):
other: Scenedesmus actus
Details on test organisms:
Scenedesmus actus was obtained from the National Institute for Environmental Studies, Japan.
initial cell density: <4.6*10^3 cells/mL
Phytoplankton cells acclimated to culture media at the exponential growth phase were incubated in 60-mL capacity polycarbonate vessels containing 60 mL of sterilized CSi culture medium (Table 2). The culture medium was then modified by changing the concentration
of phosphate (added as KH2PO4) to encourage the uptake of arsenic species.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d
Test temperature:
25 °C
pH:
7
Nominal and measured concentrations:
1.5, 7.5, 74.9, 749 and 7492 µg As/L
Details on test conditions:
Phytoplankton cells acclimated to culture media at the exponential growth phase were incubated in 60-mL capacity polycarbonate vessels containing 60 mL of sterilized CSi culture medium. The culture medium was then modified by changing the concentration of phosphate (added as KH2PO4) to encourage the uptake of arsenic species. Two different arsenic concentration treatments, high- arsenic ([As]0 = 1.0 μmol/L) and low-arsenic ([As]0 = 20 nmol/L), were used in the experimen in which As was profided in the form of NaH2AsO4.
Initially, the cell density in the culture medium was less than 4.6×10^3 cells/mL. After incubating the phytoplankton for three days, arsenic was added to the culture medium. The cultures were grown for 30 days. Phytoplankton growth was measured spectrophotometrically using a UV-VIS (ultraviolet-visible) spectrophotometer at 540 nm. The cell number was estimated with an established cell density-to-absorbance ratio. The number of cells was counted directly under a microscope. The growth rate (%) was defined by the following equation:
Growth rate[%]=(ODSample/ODControl)×100
where ODSample is the optical density of phytoplankton at 540 nm in arsenic-containing media and ODControl is the optical density of phytoplankton in arsenic-free media after seven days of cultivation.
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
75 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
50 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
215 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Reported statistics and error estimates:
EC10 have been re-calculated using Trap software (log-logistic curve fitting)
NOEC using Duncan multiple range tests
Validity criteria fulfilled:
not specified
Conclusions:
The 7 days long static toxicity test on Scenedesmus actus has yielded the following results:
NOEC (growht rate): 75 µg As / L
EC10 (growth rate): 50 µg As / L
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
7 days long chronic static toxicity test on Staurastrum paradoxum
GLP compliance:
no
Specific details on test material used for the study:
Special-grade As2O3 (AsIII) (Wako, Osaka, Japan)
Analytical monitoring:
yes
Details on sampling:
On a pre-specified day, samples were collected and filtered through 0.45-μm membrane filters under low vacuum pressure (<30 mm Hg). The filtrate sample was then stored in a cool and dark place after adding 0.50 mL of 1.0 mol/L HCl to them. After that, 20 mL of 3.0 mol/L HCl was added to the sample, and the mixture was heated on a hot plate at 100 °C for 3 h to extract the arsenic both inside and outside of the freshwater phytoplankton cells. Thereafter, the mixture volume was adjusted to 40 mL with ultrapure water, and it was also adjusted to the same concentration as that of 1.3 mol/L HCl (pH = 0).
Vehicle:
no
Details on test solutions:
CSi medium was used for the maintenance of the studied freshwater phytoplankton strains. For the growth experiment, the phosphate concentrations of the CSi medium were adjusted to 1.0 μmol/L or 50 μmol/L.
Test organisms (species):
other: Staurastrum paradoxum
Details on test organisms:
Staurastrum paradoxum was obtained from the National Institute for Environmental Studies, Japan.
initial cell density: <4.6*10^3 cells/mL
Phytoplankton cells acclimated to culture media at the exponential growth phase were incubated in 60-mL capacity polycarbonate vessels containing 60 mL of sterilized CSi culture medium (Table 2). The culture medium was then modified by changing the concentration
of phosphate (added as KH2PO4) to encourage the uptake of arsenic species.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d
Test temperature:
25 °C
pH:
7
Nominal and measured concentrations:
1.5, 7.5, 74.9, 749 and 7492 µg As/L
Details on test conditions:
Phytoplankton cells acclimated to culture media at the exponential growth phase were incubated in 60-mL capacity polycarbonate vessels containing 60 mL of sterilized CSi culture medium. The culture medium was then modified by changing the concentration of phosphate (added as KH2PO4) to encourage the uptake of arsenic species. Two different arsenic concentration treatments, high- arsenic ([As]0 = 1.0 μmol/L) and low-arsenic ([As]0 = 20 nmol/L), were used in the experimen in which As was profided in the form of NaH2AsO4.
Initially, the cell density in the culture medium was less than 4.6×10^3 cells/mL. After incubating the phytoplankton for three days, arsenic was added to the culture medium. The cultures were grown for 30 days. Phytoplankton growth was measured spectrophotometrically using a UV-VIS (ultraviolet-visible) spectrophotometer at 540 nm. The cell number was estimated with an established cell density-to-absorbance ratio. The number of cells was counted directly under a microscope. The growth rate (%) was defined by the following equation:
Growth rate[%]=(ODSample/ODControl)×100
where ODSample is the optical density of phytoplankton at 540 nm in arsenic-containing media and ODControl is the optical density of phytoplankton in arsenic-free media after seven days of cultivation.
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
749 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
670 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
3 514 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Reported statistics and error estimates:
NOEC using Duncan multiple range tests
EC10 have been re-calculated using Trap software (log-logistic curve fitting)
Validity criteria fulfilled:
not specified
Conclusions:
The 7 days long static toxicity test on Staurastrum paradoxum has yielded the following results:
NOEC (growht rate): 749 µg As/ L
EC10 (growht rate): 670 µg As/ L
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
The toxicity of As(V) to Chlorella sp. was determined using 72-h growth rate–inhibition bioassays.
GLP compliance:
no
Specific details on test material used for the study:
arsenate
Na2AsO4·7H2O, May and Baker, Dagenham, UK
Analytical monitoring:
yes
Details on sampling:
Prior to inoculation with algae, 20-ml subsamples were taken from each flask, pooled for each treatment, and stored at􏰅 18􏰉 °C for measurement of initial arsenic concentrations. Measured concentrations, not nominal, were used to calculate toxicity test endpoints
Vehicle:
no
Details on test solutions:
stock solutions of arsenate (0.2 and 15 g/L As[V]), were prepared in synthetic soft water. Synthetic soft water (70 ml) for control treatments or arsenic solutions (pH adjusted to 7.6 􏰈 0.1) were added to the flasks.
Test organisms (species):
Chlorella sp.
Details on test organisms:
Chlorella sp. 12, originally isolated from Lake Aesake, Papua New Guinea, were maintained axenically at Commonwealth Science and Industrial Research Organization, Energy Technology (Sydney, Australia). Cultures were checked microscopically monthly and plated on agar (2% bacto agar, 0.1% pepsin, and 0.1% yeast extract) several times over 12 months to ensure the absence of bacteria and other microorganisms. The algae were cultured in one-fifth strength Jaworki’s medium and incubated at 27 􏰈 1􏰉C on a 12:12 h light:dark cycle (75 􏰍mol photons/m2/ s, Philips TL 40W cool white fluorescent lighting, Caringbah, NSW, Australia).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
80-90 CaCO3/L
Test temperature:
27  °C
pH:
7.6
Nominal and measured concentrations:
between 0.75 and 60 mg As/L
Details on test conditions:
The test medium used in the bioassays was synthetic soft water (96 mg/L NaHCO , 60 mg/L CaSO ·2H O, 123 mg/L).
Each test included five arsenic concentrations and a control, each prepared in triplicate. For Chlorella sp., As(V) treatments ranged from 0.75 to 60 mg/L.
Alkalanity: 54 mg CaCO3/L

Cells in the exponential growth phase (5–6 d old) were used in bioassays after centrifugation (2,500 rpm, 7 min) and washing three times with Milli-Q water to remove residual culture medium. Flasks were inoculated with 2 to 4 􏰆 104 cells/ml and placed randomly in a growth cabinet (27 􏰈 1􏰉C, 12:12 h light:dark cycle, Philips TL 40-W cool white fluores- cent lighting, 140 􏰍mol photons/m2/s). Test flasks were rotated and shaken twice daily by hand to ensure sufficient gas exchange. The pH was recorded initially and after 72 h.

Cell density was determined daily using a Coulter multisizer IIE particle analyzer (70 mm aperture; Coulter Electronics, Luton, UK), with a correction count of background particles. The cell density data from 0 to 72 h were used to calculate the growth rate (cell division rate) of treatments by fitting a regression line to a plot of log10 (cell density) versus time (h). The slope of the plot gave the cell division rate (m) calculated as divisions per day. Growth rates for treated flasks were expressed as a percentage of the control growth rate.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
25 400 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits: 25200 - 25700 µg As/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
6 403 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Reported statistics and error estimates:
EC50 using linear interpolation method
EC10 has been been re-calculated using Trap software (log-logistic curve fitting)
Validity criteria fulfilled:
not applicable
Conclusions:
The 72-hour EC50 and EC10 values reported for growth rate of Chlorella sp. exposed to sodium arsenate dibasic are 25400 and 6403 µg As/L.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
The toxicity of As(III) to Chlorella sp. was determined using 72-h growth rate–inhibition bioassays.
GLP compliance:
no
Specific details on test material used for the study:
arsenite
NaAsO2, BDH, Poole, UK)
Analytical monitoring:
yes
Details on sampling:
Prior to inoculation with algae, 20-ml subsamples were taken from each flask, pooled for each treatment, and stored at􏰅 18􏰉 °C for measurement of initial arsenic concentrations. Measured concentrations, not nominal, were used to calculate toxicity test endpoints
Vehicle:
no
Details on test solutions:
stock solutions of arsenate (15 g/L As[III]), were prepared in synthetic soft water. Synthetic soft water (70 ml) for control treatments or arsenic solutions (pH adjusted to 7.6 􏰈 0.1) were added to the flasks.
Test organisms (species):
Chlorella sp.
Details on test organisms:
Chlorella sp. 12, originally isolated from Lake Aesake, Papua New Guinea, were maintained axenically at Commonwealth Science and Industrial Research Organization, Energy Technology (Sydney, Australia). Cultures were checked microscopically monthly and plated on agar (2% bacto agar, 0.1% pepsin, and 0.1% yeast extract) several times over 12 months to ensure the absence of bacteria and other microorganisms. The algae were cultured in one-fifth strength Jaworki’s medium and incubated at 27 􏰈 1􏰉C on a 12:12 h light:dark cycle (75 􏰍mol photons/m2/ s, Philips TL 40W cool white fluorescent lighting, Caringbah, NSW, Australia).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
80-90 CaCO3/L
Test temperature:
27  °C
pH:
7.6
Nominal and measured concentrations:
between 0.75 and 60 mg As/L
Details on test conditions:
The test medium used in the bioassays was synthetic soft water (96 mg/L NaHCO , 60 mg/L CaSO ·2H O, 123 mg/L.
Each test included five arsenic concentrations and a control, each prepared in triplicate. For Chlorella sp., As(V) treatments ranged from 0.75 to 60 mg/L.
Alkalanity: 54 mg CaCO3/L

Cells in the exponential growth phase (5–6 d old) were used in bioassays after centrifugation (2,500 rpm, 7 min) and washing three times with Milli-Q water to remove residual culture medium. Flasks were inoculated with 2 to 4 􏰆 104 cells/ml and placed randomly in a growth cabinet (27 􏰈 1􏰉C, 12:12 h light:dark cycle, Philips TL 40-W cool white fluores- cent lighting, 140 􏰍mol photons/m2/s). Test flasks were rotated and shaken twice daily by hand to ensure sufficient gas exchange. The pH was recorded initially and after 72 h.

Cell density was determined daily using a Coulter multisizer IIE particle analyzer (70 mm aperture; Coulter Electronics, Luton, UK), with a correction count of background particles. The cell density data from 0 to 72 h were used to calculate the growth rate (cell division rate) of treatments by fitting a regression line to a plot of log10 (cell density) versus time (h). The slope of the plot gave the cell division rate (m) calculated as divisions per day. Growth rates for treated flasks were expressed as a percentage of the control growth rate.
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
25 200 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits: 23300 - 29200 mg As/L
Reported statistics and error estimates:
EC50 using linear interpolation method
Validity criteria fulfilled:
not applicable
Conclusions:
The 72-hour EC50 value reported for growth rate of Chlorella sp. exposed to sodium arsenite is 25200 µg As/L.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
The toxicity of As(V) to M. arcuatum was determined using 72-h growth rate–inhibition bioassays.
GLP compliance:
no
Specific details on test material used for the study:
arsenate
Na2AsO4·7H2O, May and Baker, Dagenham, UK
Analytical monitoring:
yes
Details on sampling:
Prior to inoculation with algae, 20-ml subsamples were taken from each flask, pooled for each treatment, and stored at􏰅 18􏰉 °C for measurement of initial arsenic concentrations. Measured concentrations, not nominal, were used to calculate toxicity test endpoints
Vehicle:
no
Details on test solutions:
stock solutions of arsenate (0.2 and 15 g/L As[V]), were prepared in synthetic soft water. Synthetic soft water (70 ml) for control treatments or arsenic solutions (pH adjusted to 7.6 􏰈 0.1) were added to the flasks.
Test organisms (species):
Monoraphidium sp.
Details on test organisms:
Monoraphidium arcuatum (Kors.) Hindak, originally isolated from Lake Aesake, Papua New Guinea, were maintained axenically at Commonwealth Science and Industrial Research Organization, Energy Technology (Sydney, Australia). Cultures were checked microscopically monthly and plated on agar (2% bacto agar, 0.1% pepsin, and 0.1% yeast extract) several times over 12 months to ensure the absence of bacteria and other microorganisms. The algae were cultured in one-fifth strength Jaworki’s medium and incubated at 27 􏰈 1􏰉C on a 12:12 h light:dark cycle (75 􏰍mol photons/m2/ s, Philips TL 40W cool white fluorescent lighting, Caringbah, NSW, Australia).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
80-90 CaCO3/L
Test temperature:
27  °C
pH:
7.6
Nominal and measured concentrations:
between 0.025 and 0.4 mg As/L
Details on test conditions:
The test medium used in the bioassays was synthetic soft water (96 mg/L NaHCO , 60 mg/L CaSO ·2H O, 123 mg/L).
Each test included five arsenic concentrations and a control, each prepared in triplicate. For Chlorella sp., As(V) treatments ranged from from 0.025 to 0.400 mg/L.
Alkalanity: 54 mg CaCO3/L

Cells in the exponential growth phase (5–6 d old) were used in bioassays after centrifugation (2,500 rpm, 7 min) and washing three times with Milli-Q water to remove residual culture medium. Flasks were inoculated with 2 to 4 􏰆 104 cells/ml and placed randomly in a growth cabinet (27 􏰈 1􏰉C, 12:12 h light:dark cycle, Philips TL 40-W cool white fluores- cent lighting, 140 􏰍mol photons/m2/s). Test flasks were rotated and shaken twice daily by hand to ensure sufficient gas exchange. The pH was recorded initially and after 72 h.

Cell density was determined daily using a Coulter multisizer IIE particle analyzer (70 mm aperture; Coulter Electronics, Luton, UK), with a correction count of background particles. The cell density data from 0 to 72 h were used to calculate the growth rate (cell division rate) of treatments by fitting a regression line to a plot of log10 (cell density) versus time (h). The slope of the plot gave the cell division rate (m) calculated as divisions per day. Growth rates for treated flasks were expressed as a percentage of the control growth rate.
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
71 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
254 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits: 253 - 25 5 µg As/L
Reported statistics and error estimates:
EC50 using linear interpolation method
EC10 has been been re-calculated using Trap software (log-logistic curve fitting)
Validity criteria fulfilled:
not applicable
Conclusions:
The 72-hour EC50 and EC10 values reported for growth rate of Monoraphidium arcuatum exposed to sodium arsenate dibasic are 254 and 71 µg As/L.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
16 days long chronic As toxicity test on Botryococcus braunii
GLP compliance:
not specified
Specific details on test material used for the study:
HAsNa2O4·7H2O purchased from Himedia Laboratories Pvt. Ltd. Mumbai India
Analytical monitoring:
yes
Details on sampling:
Samples (5 mL) were withdrawn at certain time intervals and then centrifuged (Remi Instruments ltd., Mumbai India) at 5,000 g for 5 min and the supernatant fraction was analyzed for residual concentration of either As(III) or As(V) ions.
Vehicle:
no
Details on test solutions:
BG11 culture medium
Test organisms (species):
other: Botryococcus braunii
Details on test organisms:
Source: Department of Biotechnology, IIT Roorkee
inoculation: BG11 culture medium (without adding carbon source)
The algae were inoculated from the plates on another fresh agar plate after the incubation of cultures at 28 °C for 7 days in
agar plates and stored at 4 °C till required for further studies.
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
16 d
Test temperature:
28 °C
pH:
9
Nominal and measured concentrations:
nominal: 50, 100, 500, 1000, 1500 and 2000 mg As /L
Details on test conditions:
Test medium: artificial medium (BG11 medium)
Cultures were grown in an environmental chamber.
Illumination: continuous cool white fluorescent lamps at 2000 lux (Philips 40 W, cool daylight, 6,500 K) with a dark/light period of 12:12 h (75 μmol photons/m2/s).
Tests were done in duplicate.
Aeration: with 2% CO2 in air at a flow rate of approximately 150 mL/min twice in a day for 30 min by bubbling through the growth medium using an air pump.
Agitation: 20 mins.
Key result
Duration:
16 d
Dose descriptor:
EC10
Effect conc.:
217.7 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
biomass
Duration:
16 d
Dose descriptor:
EC50
Effect conc.:
16 293 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
biomass
Key result
Duration:
16 d
Dose descriptor:
EC10
Effect conc.:
2 263 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Reported statistics and error estimates:
EC10 have been re-calculated using Trap software (log-logistic curve fitting)
Validity criteria fulfilled:
not specified
Conclusions:
The 16 days long chronic toxicity test on Botryococcus braunii was resulted in the following values:
Growthrate (biomass): EC50: 16.293 mg As/L
Growthrate (biomass): EC10: 0.218 mg As/L and
Growth rate EC10: 2.263 mg As/L
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
16 days long chronic As toxicity test on Botryococcus braunii
GLP compliance:
not specified
Specific details on test material used for the study:
NaAsO2 purchased from Himedia Laboratories Pvt. Ltd. Mumbai India
Analytical monitoring:
yes
Details on sampling:
Samples (5 mL) were withdrawn at certain time intervals and then centrifuged (Remi Instruments ltd., Mumbai India) at 5,000 g for 5 min and the supernatant fraction was analyzed for residual concentration of either As(III) or As(V) ions.
Vehicle:
no
Details on test solutions:
BG11 culture medium
Test organisms (species):
other: Botryococcus braunii
Details on test organisms:
Source: Department of Biotechnology, IIT Roorkee
inoculation: BG11 culture medium (without adding carbon source)
The algae were inoculated from the plates on another fresh agar plate after the incubation of cultures at 28 °C for 7 days in
agar plates and stored at 4 °C till required for further studies.
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
16 d
Test temperature:
28 °C
pH:
9
Nominal and measured concentrations:
50, 100, 500, 1000, 1500 and 2000 mg As /L
Details on test conditions:
Test medium: artificial medium (BG11 medium)
Cultures were grown in an environmental chamber.
Illumination: continuous cool white fluorescent lamps at 2000 lux (Philips 40 W, cool daylight, 6,500 K) with a dark/light period of 12:12 h (75 μmol photons/m2/s).
Tests were done in duplicate.
Aeration: with 2% CO2 in air at a flow rate of approximately 150 mL/min twice in a day for 30 min by bubbling through the growth medium using an air pump.
Agitation: 20 mins.
Key result
Duration:
16 d
Dose descriptor:
EC10
Effect conc.:
222.4 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
biomass
Duration:
16 d
Dose descriptor:
EC50
Effect conc.:
16 705 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
biomass
Key result
Duration:
16 d
Dose descriptor:
EC10
Effect conc.:
2 263 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Reported statistics and error estimates:
EC10 have been re-calculated using Trap software (log-logistic curve fitting)
Validity criteria fulfilled:
not specified
Conclusions:
The 16 days long chronic toxicity test on Botryococcus braunii was resulted in the following values:
Growthrate (biomass): EC50: 16.705 mg As/L
Growthrate (biomass): EC10: 0.222 mg As/L and
Growth rate EC10: 2.263 mg As/L
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
not specified
Specific details on test material used for the study:
Na2HAsO4 · 7H2O, purity 98–102 %, Sigma-Aldrich, USA
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
OECD medium
Test organisms (species):
Chlorella sp.
Details on test organisms:
source: Commonwealth Science and Industrial Research Organisation (CSIRO), Lucas Heights, NSW, Australia.
Cultured: in OECD media
Stockcultures were Transferred into new growth medium every 7–14 days to ensure that the Phytoplankton populations were maintained in the exponential growth phase.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
27°C
pH:
8.1
Nominal and measured concentrations:
nominal: 0.5, 1, 2, 4, 8, 16, 32 and 64 mg As/L
Details on test conditions:
The toxicity tests followed a completely randomized design witheight treatments of As plus a control. Each treatment and control contained three replicates.
Type vessel: 25 mL polystyrene vials, Vials were acid washed and rinsed with deionised water before use.
inoculation: 3x10-4 phytoplankton cells/ml.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
9 629 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Basis for effect:
other:
Remarks:
cell density
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
3 604 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
other:
Remarks:
cell density
Reported statistics and error estimates:
EC10 and EC50 have been re-calculated using Trap software (log-logistic curve fitting)
Validity criteria fulfilled:
not specified
Conclusions:
The 72hr chronic toxicity test on Chlorella sp. was resulted in the following values for the endpoint Growth (cell density):
EC10: 3604 µg As/L
EC50: 9629 µg As/L
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
not specified
Specific details on test material used for the study:
NaAsO2, purity 90 %, Sigma-Aldrich, USA
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
OECD medium
Test organisms (species):
Chlorella sp.
Details on test organisms:
source: Commonwealth Science and Industrial Research Organisation (CSIRO), Lucas Heights, NSW, Australia.
Cultured: in OECD media
Stockcultures were Transferred into new growth medium every 7–14 days to ensure that the Phytoplankton populations were maintained in the exponential growth phase.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
27°C
pH:
8.1
Nominal and measured concentrations:
nominal: 2.0, 4.0, 8.0, 16, 32, 64, 128 and 256 mg As /L
Details on test conditions:
The toxicity tests followed a completely randomized design witheight treatments of As plus a control.Each treatment and control contained three replicates.
Type vessel: 25 mL polystyrene vials, Vials were acid washed and rinsed with deionised water before use.
inoculation: 3x10-4 phytoplankton cells/ml.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
7 787 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Basis for effect:
other:
Remarks:
cell density
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2 353 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
other:
Remarks:
cell density
Reported statistics and error estimates:
EC10 and EC50 have been re-calculated using Trap software (log-logistic curve fitting)
Validity criteria fulfilled:
not specified
Conclusions:
The 72hr chronic toxicity test on Chlorella sp. was resulted in the following values for the endpoint Growth (cell density):
EC50: 7787 µg As/L
EC10: 2353 µg As/L.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
according to guideline
Guideline:
other: ISO 2006
Principles of method if other than guideline:
72 hours long acute and chronic As toxicity test on Skeletonema costatum
GLP compliance:
not specified
Specific details on test material used for the study:
Na2(AsHO4).7H2O salt (VETEC, Rio de Janeiro)
Analytical monitoring:
yes
Details on sampling:
not specified
Vehicle:
no
Details on test solutions:
Natural coastal water
Test organisms (species):
Skeletonema costatum
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23°C
Nominal and measured concentrations:
0.12, 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 mg As/L
all measured concentrations lied close to the nominal values with a coefficient of variation ≤15 %
Details on test conditions:
Monospecific algal cells were grown for several generations in test flasks in the ISO medium.
Number of replicates per concentration: 5
Number of times the test was conducted: 2
Cell density: 10^4 cells/ml
Incubation: on shaker (100 rpm) with continuous illumination of 70mE/m2/s
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
250 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
4 460 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits: 3770 - 5150 µg As/L
Reported statistics and error estimates:
LC50 were determined by means of the Trimmed Spearman-Karber methodology; William's test (P≤0.05) was used to obtain the no-observed-effect concentration (NOEC)
Validity criteria fulfilled:
not specified
Conclusions:
The 72hr toxicity test on Skeletonema costatum was resulted in the following values for the endpoint Growth rate:
EC50: 4460 g As/L
NOEC: 250 µg As/L
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
other: Steele, R.L. and G.B. Thursby. 1983. A toxicity test using life stages of Champia parvula (Rhodophyta). In W.E. Bishop, R.D. Cardwell and B.B. Heidolph, eds., Aquatic Toxicology and Hazard Assessment: Sixth Symposium. ASTM STP 802
Principles of method if other than guideline:
The effect of arsenic on the growth and reproduction of the red alga Champia parvula was assessed in a 14-d toxicity test.
GLP compliance:
not specified
Specific details on test material used for the study:
reagent grade
Analytical monitoring:
yes
Details on sampling:
Samples for arsenite measurements were taken at test start (day 0) and before renewal of test medium (at day 7 and day 11). Samples for arsenate measurements were taken at test start (day 0).
Vehicle:
no
Details on test solutions:
Reagent-grade arsenic (as sodium arsenite or sodium arsenate) was added directly to culture flasks with digital micropipettes from a 10000 mg/L stock solution.
modified medium “f”. The medium consisted of 9.35 mg NaN03, 0.62 mg NaH2PO4.H20, 2.6 µg Fe (as Cl-) and “f116” levels of vitamins (0.06 µg BI2, 0.06 µg biotin and 12.5 µg thiamine HCl) per liter of filtered seawater (15 µm charcoal filter and 0.3 µm Balston filter). No silicate or trace metals were added to the
cultures.
Test organisms (species):
other: Champia parvula
Details on test organisms:
TEST ORGANISM
- Source: laboratory culture
- Method of cultivation: Unialgal stock cultures were maintained in 500-ml screw-capped Ehrlenmeyer flasks containing 400 ml of a modified medium “f”.

ACCLIMATION
- Culturing media and conditions: same as test

2- to 3-mm branch tips (approximately 0.025 mg dry weight) were cut to serve as inocula. Five tips were placed into each test flask. In addition, one male branch (approximately 1 cm long), visibly producing spermatia, was added to each flask. When media were changed (on days 7 and 1 1) the male branches
were clipped back to 1 cm to minimize competition with females for nutrients.
Test type:
semi-static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
14 d
Test temperature:
20-22 °C
pH:
7.8
Nominal and measured concentrations:
Test concentration were analytically confirmed.
arsenite: 0.035, 0.06, 0.095, 0.147 and 0.246 mg As/L
arsenate: between 0.1 and 10 mg As/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 ml screw-capped Ehrlenmeyer flasks
- Fill volume: 400 ml
- Renewal rate of test solution (frequency): once per week
- No. of organisms per vessel: 5 branch tips (2-3 mm)
- No. of vessels per concentration (replicates): at least 2
- No. of vessels per control (replicates): at least 2

GROWTH/TEST MEDIUM
- Standard medium used: no
- Detailed composition if non-standard medium was used: 9.35 mg NaNO3; 0.62 mg NaH2PO4 * H2O; 2.6 µg Fe (as Cl-) and "f/16" levels of vitamins (0.06 µg B12, 0.06 µg biotin, 12.5 µg thiamine * HCl) per liter of filtered seawater (15 µm charcoal filter and 0.3 µm Balston filter)

OTHER TEST CONDITIONS
- Adjustment of pH: to pH 7.8 with HCl
- Photoperiod: 16:8 h light:dark
- Light intensity and quality: 75 μE m−2 s−1 cool white fluorescent light
- Salinity (for marine algae): not specified

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Growth (final dry weight), at 14 d
- Reproduction (presence/absence of cystocarps), at 14 d

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Arsenite: SF < 2 ; Arsenate: SF=10
Key result
Duration:
14 d
Dose descriptor:
EC10
Effect conc.:
94.4 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
biomass
Remarks:
weight
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
95 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
biomass
Remarks:
weight
Duration:
14 d
Dose descriptor:
EC50
Effect conc.:
166.7 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
biomass
Remarks:
weight
Key result
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
60 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
other: reproduction
Reported statistics and error estimates:
EC10 have been re-calculated using Trap software (log-logistic curve fitting)
NOEC (growth) were determined using Tukey's test
Validity criteria fulfilled:
not specified
Remarks:
Control growth not reported
Conclusions:
The 14 days long toxicity experiment with arsenite on Champia parvula has yielded the following results:
EC10 on growth (weight): 94.4 µg As/L
NOEC value on growth (weight): 95µg As/L
NOEC value on reproduction: 60 µg As/L
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to guideline
Guideline:
other: U.S. EPA (2002)
GLP compliance:
no
Specific details on test material used for the study:
Arsenic trioxide (Merck, India (Pvt.) Ltd.)
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
Guillard’s F/2 medium
The stock toxicant solution of arsenic was prepared in Milli-Q Ultra pure water using its metallic salt of Arsenic trioxide (Merck, India (Pvt.) Ltd.).
Test organisms (species):
Skeletonema costatum
Details on test organisms:
The marine diatom, Skeletonema costatum was collected from Vellar estuary, Southeast coast of India, Parangipettai (Lat.11’290 N; and Long.79’46o E).
The species were isolated and stock culture was maintained in Guillard’s F/2 medium at Marine Algal Culture Laboratory, CAS in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipetta
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
25 °C
pH:
8.0
Salinity:
30 ppt
Nominal and measured concentrations:
15, 29, 54, 103, 195, 371 and 706 µg As/L
Details on test conditions:
All the experiments were conducted in 250 ml conical flasks with 100 ml of 3-4 days old exponentially grown algal cultures. The standard growth inhibition test procedures were followed by OECD (2002) and EPA (2002). The range finding tests were conducted for 48 hours before the definitive test.
The definitive tests were conducted in triplicate experiments using the concentrations of Arsenic viz. 15, 29, 54, 103, 195, 371 and 706 μg/l (each concentration were tested in triplicate; for 96 hours. The chronic tests were conducted in triplicate experiments using the concentrations of arsenic viz. 9, 16, 31, 58, 111, 211 and 401 μg/l (each concentration were tested in triplicate) for 120 hours. The cell density was estimated at every 24 hour intervals in all the experiments. Growth rate and percentage of growth inhibition were calculated using cell density and time (equations given below). Chlorophyll a and protein concentrations were estimated at the end of acute tests. The enzyme activities viz. Superoxide dismutase (SOD) and Malondialdehyde (MDA content) were assayed in the LOEC, Chronic concentration of arsenic (9.8, 6.9 and 112.4 μg/l) exposed for 96 hours and compared with control. During the experiment period, the physico-chemical parameters such as temperature of 25 ± 1 °C, salinity of 30‰, pH of 8.0 ± 0.3 and the light intensity of 4500 ± 500 Lux were maintained.
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
5 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Key result
Duration:
96 h
Dose descriptor:
EC10
Effect conc.:
32.6 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
112.4 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Reported statistics and error estimates:
EC10 have been re-calculated using Trap software (log-logistic curve fitting)
Validity criteria fulfilled:
yes
Conclusions:
The EC10 and EC50 values on growth rate of the species Skeletonema costatum were reported to be 32.6 and 112.4 µg As /L.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
96h hour toxicity study on Navicula sp.
GLP compliance:
no
Specific details on test material used for the study:
NaAsO2 (Shuikoushan Mining Bureau of Hengyang Inc., China)
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
modified D1 medium containing half of inorganic nutrient and one fifth of the soil nutrient extract in order to lower the ionic strength and organic matter in the medium. The modified medium with a negligible amount of dissolved organic carbon (DOC) (<1 mg /L) contains (on per liter basis) 4 mL non contaminated garden soil nutrient extract, 60 mg NaNO3, 20 mg K2HPO4, 45 mg MgSO4·7H2O, 10 mg CaCl2·2H2O, 2.5 mg ferric ammonium citrate, 50 mg Na2SiO3·9H2O, 0.1 mg MnSO4, 1 mL A5 solution which consists of 2.86 mg H3BO3, 1.86 mg MnCl2·4H2O, 0.22 mg ZnSO4·7H2O (Shanghai Xing Chemical Co., Ltd., China), 0.39 mg Na2MoO4·2H2O, 0.08 mg CuSO4·5H2O (Shanghai Zhenxin reagent factory, China) , and 0.05 mg Co(NO3)2·6H2O.

All solutions were prepared with ultrapure water (total organic carbon (TOC) <10 μg L−1) obtained from a Millipore water purification system (Millipore Synergy, USA). Arsenite stock solution (10 mM) was prepared using NaAsO2 (Shuikoushan Mining Bureau of Hengyang Inc., China).

As(III) and HA stock solutions were added to modified D1 medium to achieve the desired As(III) concentrations (0.0, 1.0, 2.0, 5.0, 10.0, 20.0, 50.0, 100.0μM) and 20 mg /L HA; an HA-free control group was also prepared (pH = 7.8 ± 0.2, Eh = 38 ± 2 mV).
Test organisms (species):
Navicula sp.
Details on test organisms:
Navicula sp. obtained from the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China) was maintained in D1 medium and grown under white incandescent light (40 μmol photons m−2 s−1, 12 h:12 h light/dark cycle) at 22±1 °C. Algae in the exponential growth phase were centrifuged (3,000 rpm, 10 min), washed twice with the modified 8 −1 medium, and then concentrated to a density of 10 cell mL .
A small volume of the concentrated algae solution was withdrawn to obtain a density of approximately 10^5 cells /mL for subsequent bioassays.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
22°C
pH:
7.8
Nominal and measured concentrations:
1.0, 2.0, 5.0, 10.0, 20.0, 50.0, and 100.0 μM
The precision of the parallel measurements was ±5 % relative standard deviation (RSD). The recovery for matrix spikes of As(III) was determined to be in the range of 90–110 %.
Details on test conditions:
Duplicate sets of solutions were shaken at 120 rpm for 48 h at 22±1 °C.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
149.8 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Remarks on result:
other: in absence of humic acids
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
149.8 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Remarks on result:
other: in presence of 20 mg/L humic acids
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
623 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Remarks on result:
other: in absence of humic acids
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
1 677 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (dissolved fraction)
Remarks:
As
Basis for effect:
growth rate
Remarks on result:
other: in presence of 20 mg/L humic acids
Reported statistics and error estimates:
EC50 using nonlinear regression analyses; NOEC were evaluated using the one-way ANOVA and paired samples t test
Validity criteria fulfilled:
not specified
Conclusions:
The 96h chronic NOEC value on growth for Navicula sp. was reported to be 149.8 µg As/L in absence or presence of humic acids.

Description of key information

Two reliable data points for acute toxicity to freshwater algae were retained for one species (Chlorella sp.) and acute effect levels ranging between 25200 µg As/L and 25400 µg As/L tested with As(III) and As(V), respectively. The lowest value is put forward for classification purposes.

Chronic toxicity data were retrieved for 9 different freshwater alga species, 7 different green algae and 2 freshwater diatoms. For the green alga Botryococcus braunii, chronic toxicity values for the endpoint growth rate (more relevant endpoint than ‘biomass’) varied between 749 and 2263 µg As/L. For Chlorella sp. a 72 h EC10 of 6403 µg As/L for the endpoint growth rate (more relevant endpoint than ‘biomass’) was further selected for PNEC derivation. For another green alga Closterium aciculare, chronic EC10 values (endpoint growth rate) varied between 4.6 (tested as AsV) and 25.5 (tested as As III) µg As/L. For the green alga Monoraphidium arcuatum a chronic 72h EC10 of 71 µg As/L (endpoint growth rate) was found. The green alga Pediastrum duplex tend to be sensitive to As V with a 7d EC10 of 5 µg As/L (endpoint growth rate). Chronic toxicity values for another green alga Scenedesmus actus revealed a 7d EC10 of 50 µg As/L (endpoint growth rate) when exposed to As V. For the green alga Staurastrum paradoxum a chronic 7d EC10 of 670 µg As/L (endpoint growth rate) was found. Toxicity data for 2 different freshwater diatoms, i.e. Achnanthidium minutissimum and Navicula sp., revealed a 7d EC10 (endpoint growth rate) of 122µg As/L for A. minutissimum and a 96h NOEC of 149.8 µg As/L.

Chronic toxicity data were retrieved for one marine diatom and 3 different marine macro-alga species. Chronic toxicity data for the marine diatom Skeletonema costatum revealed 72h-96h NOEC/EC10 values varying between 32.6 and 670 µg As/L (endpoint growth rate). For the marine red macro-alga Champia parvula, chronic toxicity values as 14 d EC10 varied between 60 µg As/L (endpoint reproduction) and 94.4 µg As/L (endpoint growth). For the marine brown macro-alga Fucus serratus, a chronic 17 weeks NOEC for the endpoint growth of 18.7 µg As/L was noted. For another marine brown macro-alga Macrocystis pyrifera, a NOEC for toxicity initiated with zoospores of 40 µg As/L (endpoint development/growth) was observed.

Key value for chemical safety assessment

EC50 for freshwater algae:
25 200 µg/L

Additional information