Strontium 3-hydroxy-4-[(1-sulfonato-2-naphthyl)azo]-2- naphthoate 5/2 hydrate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 January 2008 - 20 February 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

During the limit test singular samples for possible analysis were taken from both test groups according to the schedule below. The filter was also retained for possible analysis of the residue.

Frequency: at t=0 h, t=24 h and t=48 h
Volume: 2ml
Storage: Not applicable, samples were analysed on the day of sampling.
At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at the test substance 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.

Vehicle:

not specified

Details on test solutions:

The standard test procedures required generation of test solutions, which contained completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that would disturb the test system was prevented as much as possible (e.g. film of the test substance on the water surface).

The batch of R507-2 tested was a red powder with a purity >96.4% and not soluble in test medium at the loading rate tested.

Preparation of test solutions started with loading rates of 100 mg/I applying a 5-minute treatment period with ultrasonic waves followed by 3 days of magnetic stirring. The resulting red dispersion contained undissolved particles and a floating layer and was consequently filtered through a 0.45 µm membrane filter (Schleicher & Schuell, RC55) to remove the larger undissolved particles. The lower test concentrations for the range-finding test were prepared by dilution of the filtrate in test medium. The final test solutions were all clear and colourless.

After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, 1 ml of an algal suspension was added to each replicate providing a cell density of 10*4 cells/ml.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Species: Pseudokirchneriella subcapitata,
Strain: NIVA CHL 1 In-house laboratory culture.
Source: In-house laboratory culture.
Reason for selection: This system is an unicellular algal species sensitive to toxic substances 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.

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 10*4 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Test type:

static

Water media type:

freshwater

Total exposure duration:

48 h

Test temperature:

The temperature of the test medium was 21.7°C at the start of the test. During the exposure period the temperature measured in the incubator was maintained between 22.4 and 23.0°C.

pH:

7.8

Nominal and measured concentrations:

Analysis showed a measured concentration in the filtrate of 0.180 mg/I at the start of the test. This concentration decreased to 0.070 mg/I after 24 hours and further to 0.036 mg/I after 48 hours. The average exposure concentration was calculated to correspond to 0.08 mg/I.

Details on test conditions:

Range-finding test

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 0.45 µm filtered solution prepared at a loading rate of 100 mg/I and dilutions containing 1.0 and 10% of the filtrate.
• Three replicates were tested per concentration and three replicates in the control group.
• pH was only measured in the control and the highest test concentration.
• At the end of the test algae were not observed to verify a normal and healthy appearance.
• No sampling for determination of actual test concentrations was performed.

Limit test
Test concentrations
R507-2: A 0.45 µm filtered solution prepared at a loading rate of 100 mg/I.
Controls: Test medium without test substance or other additives.
Replicates: 6 replicates of both test groups.
2 replicates of the limit concentration without algae. 1 extra replicate of both groups for sampling purposes.

Main test:
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 720 nm using a Varian Cary 50 single beam spectrophotometer with immersion probe (pathlength =20 mm). Algal medium was used as blank.
Test vessels:100 ml, all-glass, containing 50 ml of test solution
Cell density: An initial cell density of 1 x 10*4 cells/ml.
Illumination: Continuously using TLD-lamps of the type 'Cool-white' of 30 Watt, with a light intensity within the range of 83 to 96 µE.m-2.s-1
Incubation: Vessels were distributed at random in the incubator. During incubation the algal cells were kept in suspension by continuous shaking.

Reference substance (positive control):

yes

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.08 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Details on results:

Range-finding test

Table 2 presents the percentages growth rate reduction and yield inhibition per concentration. The expected EC50 for growth rate reduction and yield inhibition exceeded the maximum solubility in test medium, i.e. was above a filtered solution prepared at 100 mg/L

All test conditions were maintained within the limits prescribed by the protocol.

Limit test

Measured test substance concentrations

At the start of the test, the measured concentration in the filtrate was 0.180 mg/I. This concentration decreased to 0.070 mg/I after 24 hours and further to 0.036 mg/I after 48 hours. The average exposure concentration was calculated to correspond to 0.08 mg/I.

Reduction of growth rate and inhibition of yield

Table 4 shows the calculation of the percentages of growth rate reduction (total test period) and the percentages of yield inhibition. Table 5 shows the calculation of the percentages of growth rate reduction at different time intervals.

No significant differences were recorded between the values for growth rate or yield between the control and the R507-2 test group.

Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.

Results with reference substance (positive control):

Algae were exposed for a period of 72 hours to K2Cr2O7 (Potassium dichromate) concentrations of 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/I and to a control. The initial cell density was 1.0 x 10*4 cells/ml.

Under the conditions of the reference study with Pseudokirchneriella subcapitata, potassium dichromate reduced growth rate of this fresh water algae species at nominal concentrations of 0.56 mg/I and higher.

The EC50 for growth rate reduction (ERC50: 0-72h) was 1.0 mg/I with a 95% confidence interval ranging from 0.70 to 1.5 mg/I. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/I. Hence, the ER C50: 0-72h for the present batch corresponds with this range.

The EC50 for yield inhibition (EvC50: 0-72h) was 0.43 mg/I with a 95% confidence interval ranging from 0.32 to 0.57 mg/I. Historical ranges are not yet available.

Table2           Percentage reduction of growth rate and inhibition of yield during the range-finding test

 

Test group* R507-2 (% filtrate)	Mean growth rate		Yield (0-48 h)
	1.1(0-48 h)	Reduction(%)	x10*4 cells/ml	Inhibition (%)
Control	0.08618		61.68
1.0	0.08782	-1.9	66.74	-8.2
10	0.08608	0.1	61.32	0.6
100	0.08595	0.3	61.31	0.6

*Test groups represent percentages of a 0.45 pm filtered solution prepared at a loading rate of 100 mg/I.

Table 3             Mean cell densities (x 10*4 cells/ml) during the limit test

 

Concentration R507-2 average (mg/I)	Exposure time (hours)
	0	24	48
Control	1.0	12.4	69.1
0.08	1.0	13.4	68.9

Table 4           Percentage reduction of growth rate (total test period) and percentage inhibition of yield during the limit test

 

Concentration R507-2 average (mg/l)	Mean growth rate		Yield (0-48h)
	JJ (0-48 h)	Reduction (%)	x10*4 cells/ml	Inhibition (%)
Control	0.08822		68.12
0.08	0.08814	0.1	67.93	0.3

 

Table 5           Percentage reduction of growth rate at different time intervals during the limit test

 

Concentration R507-2 average (mg/I)	Mean growth rate
	JJ (0-24 h)	Reduction (%)	JJ (24-48 h)	Reduction (%)
Control	0.10472		0.07172
0.08	0.10796	-3.1	0.06831	4.8

 

Table 6 shows the effect parameters based on the average exposure concentration.

 

Table 6           Effect parameters

 

Parameter	Concentration R507-2 (mg/I)
NOERC	0.08
48h-ERC50	>0.08
NOEvC	0.08
48h-EvC50	>0.08

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Pseudokirchneriel/a subcapitata, no reduction of growth rate or inhibition of yield was recorded at the maximum soluble concentration of R507-2 in test medium, which corresponded to an average measured concentration of 0.08 mg/I (NOEC).

In conclusion; both the EC50 for growth rate reduction (ER C50: 0-48h) and the EC50 for yield inhibition (EyC50: 0-48h) exceeded an average measured concentration of 0.08 mg/I.

Executive summary:

Pseudokirchneriel/a subcapitata, Fresh Water Algal Growth Inhibition Test with R507-2. The study procedures described in this report were based on the OECD guideline No. 201,

2006. In addition, the procedures were designed to meet the test methods of the EEC directive 92/69, Part C.3, 1992, the ISO International Standard 8692, 2004 and the OECD series on testing and assessment number 23, 2000.

 

The batch of R507-2 tested was a red powder with a purity > 96.4% and not soluble in test medium at the loading rate tested. The water solubility was determined to be less than 0.07 mg/I.

 

A limit test was performed based on the results of a preceding range-finding test. Exponentially growing algal cultures were exposed for 48 hours to a control and a 0.45 µm filtered solution prepared at a loading rate of 100 mg/I. The initial algal cell density was 1.0e4 cells/ml. Samples for analytical confirmation of actual exposure concentrations were taken at the start after 24 and 48 hours of exposure.

 

Analysis showed a measured concentration in the filtrate of 0.180 mg/I at the start of the test. This concentration decreased to 0.070 mg/I after 24 hours and further to 0.036 mg/I after 48 hours. The average exposure concentration was calculated to correspond to 0.08 mg/I.

 

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

 

No reduction of growth rate or inhibition of yield was recorded at the maximum soluble concentration of R507-2 in test medium, which corresponded to an average measured concentration of 0.08mg/l (NOEC).

 

In conclusion; both the EC50 for growth rate reduction (ERC50: 0-48h) and the EC50 for yield inhibition (EyC 50: 0-48h) exceeded an average measured concentration of 0.08mg/I.

Description of key information

Pseudokirchneriel/a subcapitata,Fresh Water Algal Growth Inhibition Test with R507-2. The study procedures described in this report were based on the OECD guideline No. 201,

2006. In addition, the procedures were designed to meet the test methods of the EEC directive 92/69, Part C.3, 1992, the ISO International Standard 8692, 2004 and the OECD series on testing and assessment number 23, 2000.

 

The batch of R507-2 tested was a red powder with a purity 2::96.4% and not soluble in test medium at the loading rate tested. The water solubility was determined to be less than 0.07 mg/I.

 

A limit test was performed based on the results of a preceding range-finding test. Exponentially growing algal cultures were exposed for 48 hours to a control and a 0.45 µm filtered solution prepared at a loading rate of 100 mg/I. The initial algal cell density was 10e4 cells/ml. Samples for analytical confirmation of actual exposure concentrations were taken at the start after 24 and 48 hours of exposure.

 

Analysis showed a measured concentration in the filtrate of 0.180 mg/L at the start of the test. This concentration decreased to 0.070 mg/L after 24 hours and further to 0.036 mg/L after 48 hours. The average exposure concentration was calculated to correspond to 0.08 mg/L.

 

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

 

No reduction of growth rate or inhibition of yield was recorded at the maximum soluble concentration of R507-2 in test medium, which corresponded to an average measured concentration of 0.08mg/L (NOEC).

 

In conclusion; both the EC50 for growth rate reduction (ERC50: 0-48h) and the EC50 for yield inhibition (EyC50: 0-48h) exceeded an average measured concentration of 0.08mg/L.