Reaction mass of substituted-(naphthalen-1-methyl)heteromonocyclic chloride and propane-1,2-diol

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 April 2011 to 20 May 2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The test was conducted according to the principles of GLP and a recognised ISO marine test guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Test material form:

other: Solid

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

In order to determine an appropriate test preparation method, an assessment was made of the material’s behaviour in seawater. A 1000 mg.l-1 stock was prepared in filtered seawater, and the resulting mixture was stirred for one hour. The material was poorly soluble, it was then stirred again for approximately 19 hrs, then left to settle for one hour and its behaviour assessed (SOP 402). If, the material produced floating, settled or neutrally buoyant particles or films, it was classified as poorly soluble and exposures were carried out with Water Accommodated Fractions (WAFs).
The material was characterised as poorly soluble and was therefore prepared by Water Accomodated Fractions (WAF's). WAFs were prepared by the direct addition of the required nominal weights or volumes to seawater followed by gentle stirring for approximately 20 hours and a settling period of approximately one hour. After this settling period, the middle phase of the preparation is siphoned, avoiding incorporation of undissolved particles, if present.

Test organisms

Details on test organisms:

TEST ORGANISM
- Common name:Skeletonema costatum
- Source (laboratory, culture collection): Stock Laboratory cultures
- Age of inoculum (at test initiation): 3d ±1d,
- Method of cultivation: Pre-cultures in the exponential growth phase were prepared from stock laboratory cultures by inoculating treated seawater with nutrient medium (culture medium) to a cell density of approximately 2 x 103 to 104 cells per millilitre. The pre-cultures were incubated at approximately 20±2 °C under constant illumination.

ACCLIMATION
- Culturing media and conditions (same as test or not): Same as test

Study design

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

20.1 – 21.4ºC

pH:

The pH of the control at 0h was 7.84 and at 72h ranged between 8.25 and 8.75
The pH of the test material concentrations at 0h ranged between 8.03 and 8.16, and at 72h ranged between 8.07 and 8.88

Salinity:

Culture medium is prepared from natural seawater supplied by pump from Scapa Flow, Orkney. All seawater was UV sterilised and filtered to 0.2 μm. The filtered treated seawater was then enriched with nutrients and vitamins in accordance with ISO guidelines. The salinity of the enriched natural seawater at 0h was 36‰ ± 4‰.

Nominal and measured concentrations:

The range-finding test was conducted by exposing Skeletonema costatum to a series of nominal concentration rates of 1, 10, 100 and 1000 mg/L.
Based on the results of the range-finding test the following concentration rates were assigned to the definitive test: 10, 32, 100, 320 and 1000 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml conical flasks
- Type (delete if not applicable): loosely covered with aluminium foil caps
- Material, size, headspace, fill volume: Borosilicate glass, 100 mL to which 80 mL of test medium seawater was added.
- Aeration: No
- Initial cells density: approximately 10,000 cells per ml.
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 4


GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: natural seawater supplied by pump from Scapa Flow, Orkney. All seawater was UV sterilised and filtered to 0.2 μm. The filtered treated seawater was then enriched with nutrients and vitamins in accordance with ISO guidelines.
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: Where required, adjustment carried out using either 1M HCl or NaOH and a stirring period required to ensure the pH remained constant
- Photoperiod: Constant illumination
- Light intensity and quality: 40W cool white tubes (as specified in the ISO guidelines) which were mounted at a distance of approximately 40 cm directly above the test area. Light intensity values were measured daily during the test.
- Salinity (for marine algae): Clean natural seawater with a salinity of 36‰ ± 4‰ at 0h is used in the test.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: daily by Fluorometer measurements.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: Rangefinding tests were conducted over 72h to determine the approximate concentrations at which effects were observed.
- Range finding study
- Test concentrations: Nominal concentrations: 1, 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: The Rangefinding test exhibited a 72h EC50 of 64.62mg.l-1 (Water Accommodated Fractions (WAFs)).

Reference substance (positive control):

yes

Remarks:

3,5 Dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

65.06 other: mg/L WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 61.39 - 68.10 mg/L WAF

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

32 other: mg/L WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Results with reference substance (positive control):

- Results with reference substance valid? Yes, the reference test was conducted concurrently using 3,5 Dichlorophenol at 3.2, 1.8, and 1.0mg L-1 which were prepared from a main stock of 100 mg/L. The 100 mg/L stock was stirred for a minimum of one hour, or until completely dissolved.
- EC50: 72h 2.56 mg/L, 95% Confidence limits 2.50 - 2.63 mg/L

Reported statistics and error estimates:

The raw data for each duplicate vessel and time period were averaged, to give values for each concentration of cell volume. Growth rate was calculated on the basis of these measurements. Daily intrinsic growth rate was calculated for each duplicate for each time period, using an exponential model:

Nt = No .ekt where No = volume or number at beginning of test
Nt = volume or number at time t
t = time in days
k = growth rate (.d-1)
The average value of k for each time interval was calculated for each concentration. Since the criterion of effect was the concentration causing 50% reduction in growth rate with respect to the controls, the response for each concentration was estimated from; effect = 1- (control k/treatment k)

The resulting values represent proportional reduction in growth rate. The EC50 for each time interval and the 72h EC90 and NOEC values were calculated using an appropriate statistical method from the ToxCalc Version 5 software.

Any other information on results incl. tables

Result tables see attached background information

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 72h EC50 value was 65.06 mg/ L WAF.
The 72h NOEC value was 32mg/L WAF.

Executive summary:

A study was conducted in accordance with IOS 10253 in which the marine algal species Skeletonema costatum was exposed to the test substance at test concentrations (nominal) of 10, 32, 100, 320 or 1000 mg/L for 72 hours.

The test material exhibited a 72h EC₅₀value of 65.06 mg/ L WAF (95% confidence range 61.39 - 68.10 mg/L WAF). The 72h NOEC value was 32mg/L WAF.