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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Identification: 1,3-dichloro-2-propanol
Appearance: Colourless liquid
Batch: ZMG-197685
Purity/Composition: 99.2%
Test item storage: At room temperature container flushed with nitrogen
Stable under storage conditions until: 29 December 2018 (expiry date)
Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Frequency at t=0 h, t=24 h and t=72 h
Volume 9.0 mL
Storage Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
At the end of the exposure period, the replicates with algae were not pooled before sampling. Instead, samples were taken from replicate A of each test group.
Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate item concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
Additionally, reserve samples of 9.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.
Vehicle:
yes
Details on test solutions:
The batch of 1,3-dichloro-2-propanol tested was a colourless liquid with a purity of 99.2% and the item was completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with the highest concentration of 100 mg/L. 73.5 µL of test item was introduced into medium using a pipette, under nitrogen in a glovebox. A 15 – 22 minute period of magnetic stirring was applied to accelerate dissolution of the test item in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
After preparation, volumes of 40 mL were added to each replicate of the respective test concentration. Subsequently, 0.8 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL.
Any residual volumes were discarded.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Species Pseudokirchneriella subcapitata, strain: NIVA CHL 1
Source In-house laboratory culture.
Reason for selection This system is an unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.
Stock culture Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
Light intensity 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
Stock culture medium M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:
NaNO3 500 mg/L
K2HPO4.3H2O 52 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3.10H2O 54 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L
Pre-culture 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
Pre-culture medium Adjusted M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition:
NH4Cl 15 mg/L
MgCl2.6H2O 12 mg/L
CaCl2.2H2O 18 mg/L
MgSO4.7H2O 15 mg/L
KH2PO4 1.6 mg/L
FeCl3.6H2O 64 µg/L
Na2EDTA.2H2O 100 µg/L
H3BO3 185 µg/L
MnCl2.4H2O 415 µg/L
ZnCl2 3 µg/L
CoCl2.6H2O 1.5 µg/L
CuCl2.2H2O 0.01 µg/L
Na2MoO4.2H2O 7 µg/L
NaHCO3 300 mg/L
Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)
HEPES buffer 6 mmol/L
pH 7.1 ± 0.3
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Details on test conditions:
A range-finding test was performed to provide information about the range of concentrations to be used in the final test. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
• Exponentially growing algal cultures were exposed to a range of 0.10 to 100 mg/L increasing by a factor of 10 and to a control.
• Three replicates were tested per concentration and three replicates in the control group.
• One extra test vessel per concentration without algae was used as background for the determination of the algal cell density at each time interval.
• pH was only measured in the control and the highest test concentration.
• Cell densities were determined only at the end of the exposure period.
Final Test
Test Concentrations
1,3-dichloro-2-propanol 10, 18, 32, 56 and 100 mg/L
Controls Test medium without test item or other additives.
Replicates 3 replicates of each test concentration,
6 replicates of the control,
1 extra replicate of each test group for sampling purposes,
1 or 2 replicates of each test concentration without algae.

Test Procedure and Conditions
Test duration 72 hours
Test type Static
Test vessels 40 mL, airtight closed, with no headspace, containing 40 mL of test solution
Medium Adjusted M2
Cell density An initial cell density of 1 x 104 cells/mL.
Illumination Continuously using TLD-lamps with a light intensity within the range of 73 to 76 µE.m-2.s-1.
Incubation Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

Measurements and Recordings
pH At the beginning and at the end of the test.
Temperature of medium Continuously in a temperature control vessel.
Appearance of the cells At the end of the final test microscopic observations were performed on 100 mg/L to observe for any abnormal appearance of the algae.

Recording of Cell Densities
At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with cuvettes (path length =10 mm). Algal medium was used as blank and the extra replicates, without algae, as background for the treated solutions
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
NOEC
Remarks:
based on statistical significance
Effect conc.:
18 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Remarks:
based on biological relevance
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Remarks:
based on statistical significance
Effect conc.:
18 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
NOEC
Remarks:
based on biological relevance
Effect conc.:
56 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
>= 41 - <= 78 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Validity criteria fulfilled:
yes
Conclusions:
In conclusion, under the conditions of the present study with Pseudokirchneriella subcapitata, no biological relevant inhibition of growth rate was recorded at any of the concentrations of 1,3-dichloro-2-propanol tested.
The EC50 values for growth rate and yield inhibition (72h-ERC50 and 72h-EYC50, respectively) were beyond the range tested.
The 72h-NOEC for growth rate inhibition was 100 mg/L based on biological relevance and 18 mg/L based on statistical significance.
The 72h-NOEC for yield inhibition was 56 mg/L based on biological relevance and 18 mg/L based on statistical significance.
Executive summary:

The objective of the study was to evaluate 1,3-dichloro-2-propanol for its ability to generate toxic effects in Pseudokirchneriella subcapitata during an exposure period of 72 hours and, if possible, to determine the NOEC, EC10and EC50for both inhibition of growth rate and inhibition of yield.

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2000.

The batch of 1,3-dichloro-2-propanol tested was a colourless liquid with a purity of 99.2% and the item was completely soluble in test medium at the concentrations tested.

A final test was performed based on a preceding range-finding test. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to 10, 18, 32, 56 and 100 mg 1,3-dichloro-2-propanol per litre. The initial algal cell density was 104cells/mL.The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure. Exposure took place in airtight closed vessels with headspace reduced to a minimum.

Samples taken from the highest test concentration was analysed. The measured concentrations were at the level of nominal throughout the test (i.e. 88-101%). Based on these results, effect parameters were expressed in terms of analytically confirmed nominal concentrations.

Statistically significant inhibition of growth rates and yield was found at the three highest test concentrations. However, only the inhibition of yield at the highest concentration was biologically relevant (i.e. >10%).

The study met the acceptability criteria prescribed by the study plan and was considered valid.

In conclusion, under the conditions of the present study withPseudokirchneriella subcapitata, no biological relevant inhibition of growth rate was recorded at any of the concentrations of 1,3-dichloro-2-propanol tested.

Description of key information

Under the conditions of the present study with Pseudokirchneriella subcapitata, no biological relevant inhibition of growth rate was recorded at any of the concentrations of 1,3-dichloro-2-propanol tested.

The EC50 values for growth rate and yield inhibition (72h-ERC50 and 72h-EYC50, respectively) were beyond the range tested.

The 72h-NOEC for growth rate inhibition was 100 mg/L based on biological relevance and 18 mg/L based on statistical significance.

The 72h-NOEC for yield inhibition was 56 mg/L based on biological relevance and 18 mg/L based on statistical significance.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
100 mg/L

Additional information