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Ecotoxicological information

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
Study period:
30-Apr-2010 to 11-May-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
other: analytical methods
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
swissmedic: Date of inspection: 05 to 09-Nov-2007 and 26 to 30-Nov-2007; Date of decision: 2008-04-30, Date of signature: 12-Nov-2008
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable
Analytical monitoring:
yes
Details on sampling:
For measurement of the actual concentrations of the test item, duplicate samples were taken from the test media of all treatments at the start of the test (without algae) and at the end of the test (containing algae).

For sampling at the end of the test, the test medium of the treatment replicates was pooled.

All samples were stored deep-frozen (at about -20 °C) immediately after sampling until analysis. Based on a pre-experiment (non-GLP), the test item proved to be sufficiently stable in the test water under these storage conditions.

The concentration of the test item Sodiumtrimetaphosphate was determined in the duplicate test medium samples from the nominal test concentration of 100 mg/L. The samples from all lower test concentrations were not analyzed, since these concentrations were below the NOEC determined in this test. From the control samples, one of the duplicate samples was analyzed from the corresponding sampling times.
The analytical procedure and results are described in the corresponding attached APPENDIX I – ANALYTICAL INVESTIGATIONS.
Vehicle:
no
Details on test solutions:

Material
50 mL Erlenmeyer flasks were used per replicate containing 15 mL of test solution. Each test flask was covered with a glass dish. The test flasks were labeled with the study number and all necessary additional information to ensure unique identification. During exposure, the test solutions were continuously stirred by magnetic stirrers.

Experimental Conditions
The test flasks were incubated in a temperature-controlled water bath at a temperature of 22 °C and illuminated by fluorescent tubes (Philips TLD 36W-1/840), installed above the test flasks.
The test flasks were positioned randomly and repositioned daily. The mean measured light intensity at the level of the test solutions was approximately 7300 Lux (range: 6490 to 7980 Lux, measured at nine places in the experimental area). The light intensity over the incubation area was within ±15% from the average light intensity as recommended by the guideline.

Study Design
The selection of the test concentrations was based on the results of a range-finding test (non-GLP).

The following nominal concentrations of the test item were tested: 1.0, 3.2, 10, 32 and 100 mg/L. Additionally, a control was tested in parallel (test water without test item). Nominal concentrations of the test item exceeding 100 mg/L were not tested, in accordance with the test guidelines.

The test design included three replicates per test concentration and six replicates of the control.

The test was started using a nominal algal cell density of 10000 cells/mL. The initial cell density was selected according to the recommendations of the OECD test guideline. The algal cell density in the pre-culture was determined by an electronic particle counter (Coulter Counter®, Model ZM).

A static test design was applied. The duration of the test was 72 hours.

Dosage
The test medium of the highest nominal concentration of 100 mg/L was prepared by dissolving 35.56 mg of the test item completely in 355 mL of test water under intense stirring for 15 minutes at room temperature. The test medium of the highest test concentration was diluted with test water to prepare the test media of the lower test concentrations. The test media were prepared just before the start of the test.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test organism used for the study was Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum), Strain No. 61.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany). The algae were cultivated at Harlan Laboratories under standardized conditions according to the test guidelines.

Nygaard et al. recommended describing the taxa within the Genus Raphidocelis HINDAK as:

Raphidocelis subcapitata (KORSIKOV) nov. comb.
Basionym: Ankistrodesmus subcapitatus KORSIKOV
Syn.: Kirchneriella subcapitata KORSIKOV
Syn.: Selenastrum capricornutum PRINTZ
Syn.: NIVA-CHL 1

An inoculum culture was set up three days before the start of the exposure. The algae were cultivated under the test conditions and were kept in the exponential growth phase until inoculation of the test solutions.

For evaluation of the algal quality and experimental conditions, potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions. The result of the latest positive control test performed in March 2010 showed that the sensitivity of the test system was within the internal historical range (72-hour EC50 for the growth rate: 0.94 mg/L (study C86922), range of the 72-hour EC50 for the growth rate from 2000 to 2010: 0.71 1.7 mg/L).

The test method and the test species are recommended by the test guidelines.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
not applicable
Hardness:
The water hardness (calculated) of the test water was 0.24 mmol/L (= 24 mg/L as CaCO3).
Test temperature:
The test flasks were incubated in a temperature-controlled water bath at a temperature of 22 °C.
pH:
At the start of the test, the pH measured in the treatments was between 8.0 and 8.1. At the end of the test, pH values of 8.1 to 8.2 were measured.
Dissolved oxygen:
not applicable
Salinity:
According to OECD Test guideline
Nominal and measured concentrations:
The nominal concentrations of the test item of 1.0, 3.2, 10, 32 and 100 mg/L were tested. Additionally, a control was tested in parallel (test water without test item).

The measured concentration of the test item in the test medium of the test concentration of 100 mg/L was 100 and 89% of the nominal value at the start and at the end of the test, respectively. Thus, the correct dosing of the test item Sodiumtrimetaphosphate at test start was confirmed. Furthermore, it has been demonstrated that the test item was sufficiently stable during the test period of 72 hours under the conditions of the test, and the reported biological results are based on the nominal concentrations of the test item.

Details on test conditions:
Reconstituted test water prepared according to the test guidelines was used for algal cultivation
and testing. For further informations please see section - any other information on materials and methods oncl. tables.

50 mL Erlenmeyer flasks were used per replicate containing 15 mL of test solution. Each test flask was covered with a glass dish. The test flasks were labeled with the study number and all necessary additional information to ensure unique identification. During exposure, the test solutions were continuously stirred by magnetic stirrers.

The test flasks were incubated in a temperature-controlled water bath at a temperature of 22 °C and illuminated by fluorescent tubes (Philips TLD 36W-1/840), installed above the test flasks. The test flasks were positioned randomly and repositioned daily. The mean measured light intensity at the level of the test solutions was approximately 7300 Lux (range: 6490 to 7980 Lux, measured at nine places in the experimental area). The light intensity over the incubation area was within ±15% from the average light intensity as recommended by the guideline.
Reference substance (positive control):
yes
Remarks:
twice a year
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % CL not reported
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % CL not reported
Details on results:
The measured concentration of the test item in the test medium of the test concentration of 100 mg/L was 100 and 89% of the nominal value at the start and at the end of the test, respectively (see analytical results and Table 2 in the corresponding appendix). Thus, the correct dosing of the test item

Sodiumtrimetaphosphate at test start was confirmed. Furthermore, it has been demonstrated that the test item was sufficiently stable during the test period of 72 hours under the conditions of the test, and the reported biological results are based on the nominal concentrations of the test item.

The influence of the test item on the growth of the algae is shown in the attached Table 1 to Table 3 and in Figure 1.

The test item had no statistically significant inhibitory effect on the growth of the algae (average growth rate and yield) during the test period of 72 hours up to and including the highest nominal test concentration of 100 mg/L (results of Williams t-tests, one-sided smaller, α = 0.05, Table 2 and Table 3).

The test concentration of 100 mg/L was, therefore, determined to be the 72-hour NOEC. The NOEC might even be higher, but concentrations of the test item exceeding 100 mg/L were not tested, in accordance with the test guidelines. The 72-hour LOEC was higher than 100 mg/L.

The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal test concentration of 100 mg/L and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration.

Thus, it can be concluded that the test item Sodiumtrimetaphosphate has no acute toxic effect on the growth of the freshwater green algal species Pseudokirchneriella subcapitata up to a concentration of 100 mg/L under the present test conditions.

In the control the biomass increased by a factor of 113 over 72 hours (Table 1). The validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates in the control (section-by-section growth rates, Table 4) during 72 hours was 8.2%. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35%. Thus, the validity criterion was fulfilled.

The coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 1.6%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled.

No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.

At the start of the test, the pH measured in the treatments was between 8.0 and 8.1. At the end of the test, pH values of 8.1 to 8.2 were measured (Table 5). The water temperature during the test was 22 °C.
Results with reference substance (positive control):
For evaluation of the algal quality and experimental conditions, potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions. The result of the latest positive control test performed in March 2010 showed that the sensitivity of the test system was within the internal historical range (72-hour EC50 for the growth rate: 0.94 mg/L (study C86922), range of the 72-hour EC50 for the growth rate from 2000 to 2010: 0.71 1.7 mg/L).
Reported statistics and error estimates:
The average growth rate over the test duration and the section-by-section growth rates (daily growth rates between the sampling times) were calculated.
Growth rate and yield were calculated for each test flask. The mean values for growth rate and yield were calculated for each treatment. The tabulated values represent rounded results obtained by calculation using the exact raw data.

The 72-hour EC10, EC20and EC50 values of the test item could not be determined due to the absence of a significant inhibitory effect of the test item on the algal growth at the tested concentrations.

For the determination of the LOEC and NOEC, average growth rate and yield at the test concentration of 100 mg/L were compared to the control values by Williams t - tests.

Please see attatched tables 1 to 5 for Table 1 Biomass of Algae, Table 2 Average Growth Rates (μ), Table 3 Yield (Y), Table 4 Section-by-Section Growth Rates & Table 5 pH Values in the Treatments.

Please also see attached Figure 1 Growth Curves of the Algae over the Test Duration of 72 Hours.

Validity criteria fulfilled:
yes
Conclusions:
The test item Sodiumtrimetaphosphate has no acute toxic effect on the growth of Pseudokirchneriella subcapitata up to a concentration of 100 mg/L under the present test conditions.
This study is conducted according to the appropriate guidelines (EU ) and under the conditions of GLP and therefore the study is considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement as a key study for this endpoint.
Executive summary:

The influence of the test item Sodiumtrimetaphosphate on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to OECD Guideline 201 (2006), the EU Commission Directive 92/69/EEC, C.3 (1992) and the Commission Regulation (EC) No 440/2008, C.3).

 

The nominal concentrations of the test item of 1.0, 3.2, 10, 32 and 100 mg/L were tested. Additionally, a control was tested in parallel (test water without test item).

 

The measured concentration of the test item in the test medium of the test concentration of 100 mg/L was 100 and 89% of the nominal value at the start and at the end of the test, respectively. Thus, the correct dosing of the test item Sodiumtrimetaphosphate at test start was confirmed. Furthermore, it has been demonstrated that the test item was sufficiently stable during the test period of 72 hours under the conditions of the test, and the reported biological results are based on the nominal concentrations of the test item.

 

The test item had no statistically significant inhibitory effect on the growth of the algae (average growth rate and yield) during the test period of 72 hours up to and including the highest nominal test concentration of 100 mg/L.

 

The test concentration of 100 mg/L was, therefore, determined to be the 72 hour NOEC. The NOEC might even be higher, but concentrations of the test item exceeding 100 mg/L were not tested, in accordance with the test guidelines. The 72‑hour LOEC was higher than 100 mg/L.

 

Thus, it can be concluded that the test item Sodiumtrimetaphosphate has no acute toxic effect on the growth of Pseudokirchneriella subcapitata up to a concentration of 100 mg/L under the present test conditions.

Description of key information

One key study exists. This study had been conducted in accordance with a recommended guideline (OECD 201, EU Method C.3) and under the conditions of GLP. As such, no further testing is considered necessary.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
100 mg/L

Additional information

On the basis of a limit test, the NOEC based on growth inhibition of trisodium trimetaphosphate has been determined to be >100 mg/l.