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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:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. Conclusive valid guideline study under GLP conditions.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EC) No 761/2009 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), 2009, C.3: Algal
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. QA statement)
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 test item concentrations, duplicate samples were taken from the test media of all test concentrations at the start of the test (without algae) and at the end of the test (containing algae). At the same sampling times, duplicate samples were also taken from the control.

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

The samples were analyzed immediately after sampling. The concentrations of the test item Dilauroyl peroxide were determined in the duplicate test medium samples from all treatments.

The analytical procedure and results are described in the attached Appendix I - Analytical Investigations.
Vehicle:
no
Details on test solutions:
Due to the low water solubility of the test item (0.06 mg/L, see study MIRA20090934-water solubility, AkzoNObel), dispersions of the test item were prepared. No auxiliary solvent or emulsifier was used.

Before weighing in the test item, the test item flakes were pestled.

The dispersion of the test item with the loading rate of 1.0 mg/L was prepared by dispersing 3.6213 mg of the test item in 3110 mL of test water. The dispersion of the test item with the loading rate of 100 mg/L was prepared by dispersing 310.27 mg of the test item in 3100 mL of test water. Both dispersions were intensely stirred for 72 hours at room temperature in the dark. During stirring the flasks were stoppered with glass stoppers and the air-space was kept at a minimum to avoid test item losses due to volatilization.

The stirring period of 72 hours was chosen based on study MIRA20090934-water solubility, to dissolve a maximum amount of test item in the test water.

After the stirring period of 72 hours, undissolved test material was separated from the test water (as far as possible) using a separation funnel. The test item could not be filtered due to potential absorption of the test item to filter materials. Thus, the dispersions were left in the separation funnel to separate for about 30 minutes and the middle layers were used as test water.

The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

Half of the volume of the test media was immediately used for test part 1. For test part 2, the remaining volumes of test media were left for ageing over a period of 7 days in a closed system to allow the formation of the eventual degradation products. Test part 2 was performed with the 7-day old test media.

The control medium (test water without test item) for test part 2 was also left for ageing over a period of 7 days in a closed system.
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.
Test type:
static
Water media type:
freshwater
Limit test:
yes
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 water temperature during the test was maintained between 22 and 23 °C.
pH:
To keep the pH of the test media as constant as possible 6 mmol/L HEPES-buffer (corresponding to 1430 mg/L) were added to the test water.At the start of the test (test part 1), the pH measured in the treatments was 8.0 to 8.1. At the end of test part 1, pH values of 8.7 to 9.7 were measured (for further information please see attached Table 5). The increase of the pH during the test was caused by the uptake of CO2 by the algae due to their rapid growth, despite the test media being stirred during the test.
Dissolved oxygen:
not applicable
Salinity:
according to OECD guideline
Nominal and measured concentrations:
At the start of the test, the analytically determined concentrations of Dilauroyl peroxide in the test media of the loading rates of 1.0 and 100 mg/L were 0.235 and 2.12 mg/L, respectively (for further information please see Appendix I - Analytical investigations). Thus, the measured concentrations were above the water solubility limit of the test item (0.06 mg/L; study MIRA20090934-water solubility, AkzoNobel) due to undissolved test material in the test media. During the test period of 72 hours, the test item concentrations in the test media decreased. At the end of the test, the measured concentrations were 0.034 and 0.52 mg/L, respectively.

For further information on measured test item concentrations please see section " any other information on materials and methods incl. tables", below.

The mean measured test item concentrations were above the water solubility limit (0.06 mg/L, see study MIRA20090934-water solubility, AkzoNobel).
Details on test conditions:
Reconstituted test water prepared according to the test guidelines was used for algal cultivation and testing. For further information on test water please see section" Any other information on materilas and methods incl. tables"

As the test item was assumed to volatilize from water (Vapour pressure at 20 °C: 1.6 x 10-4 Pa according to Akzo Document n° “2.420.007 –“, resulting Henry constant based on a water solubility of 0.06 mg/L: 1.06 Pa.m3/mol), glass stoppered 50 mL Erlenmeyer flasks were used (closed system) completely filled with about 60 mL of test medium, minimizing the air-space in the flasks and avoiding potential losses of test item. 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 to 23 °C (test parts 1 and 2) 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 7000 Lux (range: 6530 to 7560 Lux) in the first part of the test and approximately 6600 Lux (range: 6100 to 7180 Lux) in the second part of the test (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.

At request of the Sponsor, the test consisted of two parts: the test item Dilauroyl peroxide was tested in part 1; eventual degradation products of the test item were tested in part 2.

A limit test was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effects on the algae up to its limit of solubility in the test water. Thus, a saturated solution of the test item was tested.

Therefore, test water was loaded with 1.0 and 100 mg/L. Both loading rates resulted in measured concentrations above the water solubility limit of the test item (0.06 mg/L, see study MIRA20090934-water solubility, AkzoNobel). The loading rate of 100 mg/L was tested at request of the Sponsor for registration purposes. Control groups (test water without test item) were tested in parallel.
The test design included six 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).
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.089 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
and yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
and yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
and yield
Details on results:
Influence of the test item Dilauroyl peroxide on algal growth (test part 1):
At the start of the test, the analytically determined concentrations of Dilauroyl peroxide in the test media of the loading rates of 1.0 and 100 mg/L were 0.235 and 2.12 mg/L, respectively. Thus, the measured concentrations were above the water solubility limit of the test item (0.06 mg/L; study MIRA20090934-water solubility, AkzoNobel) due to undissolved test material in the test media. During the test period of 72 hours, the test item concentrations in the test media decreased. At the end of the test, the measured concentrations were 0.034 and 0.52 mg/L, respectively.

Please find more detailed information on the mean measured test item concentrations (calculated as the geometric means of the concentrations measured at the start and at the end of the test (test part 1)) under "any other information on materials and methods incl. tables" above.

The mean measured test item concentrations were above the water solubility limit (0.06 mg/L, see study MIRA20090934-water solubility, AkzoNobel).
The biological results were related to the loading rates and to the mean measured test item concentrations.

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 at the loading rate of 1.0 mg/L, corresponding to a mean measured test item concentration of 0.089 mg/L (result of Welch t-test, one-sided smaller, alpha = 0.05). The growth inhibiting effects at the loading rate of 100 mg/L were statistically significant.

The loading rate of 1.0 mg/L, corresponding to a mean measured concentration of 0.089 mg/L, was, therefore, determined to be the 72 hour NOEC. The 72 hour NOEC based on the mean measured concentration lies above the water solubility limit of the test item (0.06 mg/L, see study MIRA20090934-water solubility, Akzo Nobel).

The 72 hour LOEC was determined to be at the loading rate of 100 mg/L (with 16% inhibition of yield and 3.5% inhibition of average growth rate). In the pre-test (non-GLP), no inhibiting effects could be observed at this loading rate.

For a summary of the biological results please see section "any other information on results incl. tables".

The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing in the test item treated medium (loading rate 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.

Thus, it can be concluded that the test item Dilauroyl peroxide has no toxic effects on the growth of the freshwater green algal species Pseudokirchneriella subcapitata up to its water solubility limit under the conditions of the test.

In the control the biomass increased by a factor of 123 over 72 hours (see attached 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, see attached Table 4) during 72 hours was 16%. 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 2.7%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled.

Concerning the appearance of the test media, inhomogeneous dispersed test item was observed in the test media of the test item treatments throughout the test period.

At the start of the test (test part 1), the pH measured in the treatments was 8.0 to 8.1. At the end of test part 1, pH values of 8.7 to 9.7 were measured (see attached Table 5). The increase of the pH during the test was caused by the uptake of CO2 by the algae due to their rapid growth, despite the test media being stirred during the test. The water temperature during the test was maintained between 22 and 23 °C.


Influence of eventual degradation products of the test item on algal growth (test part 2):
At the start of test part 2, the analytically determined concentrations of Dilauroyl peroxide in the test media of the loading rates of 1.0 and 100 mg/L were 0.0673 and 0.228 mg/L, respectively (for further information please see Appendix I - Analytical Investigations). During the test period of 72 hours, the test item concentrations in the test media decreased. After 72 hours of test duration, the measured concentrations were below LOQ (Limit of quantification, LOQbio: 0.02 mg/L).

For further details on the mean measured test item concentrations (calculated as the geometric means of the concentrations measured at the start and at the end of the test (test part 2)) please see section "any other information on materials and methods incl. tables", above.

The biological results were related to the loading rates of the test item.

The influence of the test item on the growth of the algae is shown in the attached Table 6 to Table 8 and in Figure 2.

The test item had no statistically significant inhibitory effect on the yield of the algae during the test period of 72 hours at the loading rate of 1.0 mg/L (result of Williams t-test, one-sided smaller, alpha = 0.05, Table 8). The parameter “average growth rate” was statistically significantly inhibited in both test item treatments (results of Welch t-test, one-sided smaller, alpha = 0.05, Table 7). However, the inhibition of the growth rate of 1.2% at the loading rate of 1.0 mg/L was not seen as a biologically relevant toxic effect. Thus, the overall 72-hour NOEC was determined to be at the loading rate of 1.0 mg/L.

The loading rate of 100 mg/L was determined to be the 72-hour LOEC (with 24% inhibition of yield and 6.0% inhibition of average growth rate). In the pre-test (non-GLP), no inhibiting effects could be observed at this loading rate (see Appendix II).

For a summary of the biological results (on the basis of loading rates of the test item) please see section " any other information on results incl. tables".

The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing in test item treated media and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item.

Thus, it can be concluded that the test item Dilauroyl peroxide and its eventual degradation products have no toxic effects on the growth of the freshwater green algal species Pseudokirchneriella subcapitata up to its water solubility limit under the conditions of the test.

In the control the biomass increased by a factor of 84 over 72 hours (for further information please see attached Table 6). 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, attached Table 4) during 72 hours was 33%. 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 1.0%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled.

The test media were inhomogeneous dispersions of the test item throughout the test period

At the start of the test (test part 2), the pH measured in the treatments was between 8.0 and 8.1. At the end of the test (test part 2), pH values of 8.8 to 10.2 were measured (for further information please see attached Table 10). The increase of the pH during the test was caused by the uptake of CO2 by the algae due to their rapid growth, despite the test media being stirred during the test. The water temperature during the test was maintained at 23 °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 forthe growth rate from 2000 to 2010: 0.71 to 1.7 mg/L).
Reported statistics and error estimates:
The 72-hour EC10, EC20 and EC50 values for the inhibition of average growth rate and yield and their 95% confidence intervals could not be calculated. The EC50- and (as far as possible) the EC20-values were determined directly from the raw data.

For the determination of the LOEC and NOEC, average growth rate and yield at the test concentrations were compared to the control values by Williams t-test or Welch t-test.

Two outliers, one in each test part (part 1 and 2), were identified by the outlier test after Dixon and Hartley. The outliers were excluded from the data evaluation (see attached Table 1 and Table 6).

The biological results /test part one) can be summarized as follows (on the basis of loading rates and mean measured test item concentrations):

 

Parameter

Growth rate

Yield

Growth rate

Yield

(0-72 h)

(based on loading rates)

(based on mean measured concentrations)

 

(mg/L)

(mg/L)

EC10  

>100

n.d.

>1.0

n.d.

EC20  

>100

>100

>1.0

>1.0

EC50  

>100

>100

>1.0

>1.0

NOEC 

1.0

1.0

0.089

0.089

LOEC 

100

100

1.0

1.0

n.d.:       could not be determined

The biological results (test part two) can be summarized as follows (on the basis of loading rates of the test item):

 

Parameter

Growth rate

Yield

(0-72 h)

EC10  (mg/L)

>100

n.d.

EC20  (mg/L)

>100

n.d.

EC50  (mg/L)

>100

>100

NOEC (mg/L)

1.0

1.0

LOEC (mg/L)

100

100

         n.d.:            could not be determined

 

Validity criteria fulfilled:
yes
Conclusions:
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 at the loading rate of 1.0 mg/L, corresponding to a mean measured test item concentration of 0.089 mg/L (result of Welch t-test, one-sided smaller, alpha = 0.05). The growth inhibiting effects at the loading rate of 100 mg/L were statistically significant.
Executive summary:

The influence of the test item Dilauroyl peroxide and its eventual degradation products on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in 72‑hour static tests 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 test item Dilauroyl peroxide was dispersed in test water at loading rates of 1.0 and 100 mg/L. The dispersions were stirred for 72 hours in order to dissolve a maximum amount of the test item in the test water. After the stirring period, the dispersions were let to settle in a separation funnel and the middle layers were used as test media.The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

 

Half of the volumes of the test media were used immediately to examine the influence of the test item Dilauroyl peroxide on the algal growth (test part 1). The remaining volumes of the test media were left in a closed system for ageing over a period of 7 days in order to allow the test item to degrade and to test the influence of eventual degradation products of the test item on the algal growth (test part 2). The test duration of both tests was 72 hours.

 

Control groups were run in parallel. The control medium for the degradation products test (test part 2) was left in a closed system for ageing over a period of 7 days.

 

At the start of the Dilauroyl peroxide parent test (test part 1)test item concentrationsof0.24 and 2.1 mg/Lwere measured in the test media with the loading rates of 1.0 and 100 mg/L, respectively. Thus, the measured concentrations were above the water solubility limit of the test item(0.06 mg/L; study MIRA20090934-water solubility, Akzo Nobel) due to undissolved test material in the test media. During the test period of 72 hours, the test item concentrations in the test media decreased. At the end of the test, the measured concentrations were 0.034 and 0.52 mg/L,respectively.

 

The mean measured test item concentrations were calculated as the geometric means of the concentrations measured at the start and at the end of the test(test part 1):

 

Loading rate
(mg/L)

Mean measured concentration
(
mg/L)

1.0

0.089

100

1.0

 

 

The biological resultsof the Dilauroyl peroxide parent test (test part 1)were related to the loading rates and to the mean measured concentrations of the test item.The biological resultsof the degradation products test (7-days aged test media, test part 2)were related to the loading rates of the test item.

 

The biological results of theDilauroyl peroxide parent test (test part 1) were as follows (based on the loading rates and mean measured concentrations of the test item):

 

Parameter

Growth rate

Yield

Growth rate

Yield

(0-72 h)

(based on loading rates)

(based on mean measured concentrations)

 

(mg/L)

(mg/L)

EC10  

>100

n.d.

>1.0

n.d.

EC20  

>100

>100

>1.0

>1.0

EC50  

>100

>100

>1.0

>1.0

NOEC 

1.0

1.0

0.089

0.089

LOEC 

100

100

1.0

1.0

n.d.:       could not be determined

 

 

The biological results of the degradation products test(7-days aged test media, test part 2) were as follows (based on the loading rates of the test item):

 

Parameter

Growth rate

Yield

(0-72 h)

EC10  (mg/L)

>100

n.d.

EC20  (mg/L)

>100

n.d.

EC50  (mg/L)

>100

>100

NOEC (mg/L)

1.0

1.0

LOEC (mg/L)

100

100

         n.d.:            could not be determined

 

 

As the water solubility of the test item was determined to be 0.06 mg/L (study MIRA20090934-water solubility, Akzo Nobel) it can be concluded that the test item Dilauroyl peroxide and its degradation products have no biological relevant toxic effects on the growth of Pseudokirchneriella subcapitata up to the test item’s water solubility limit under the conditions of the test.

 

Description of key information

One valid algae study carried out in a closed system is available. No effects were found using the WAF method up to 100 mg/L corresponding to the limit of solubility or higher.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
100 mg/L

Additional information

2 studies on algae are available, one invalid study (Gancet, 2008b) which was performed using filtered stock solutions, perhaps removing any dissolved substance present, and a reliable GLP study on Pseudokirchneriella subcapitata following the 2006 updated OCED 201 (Weber, 2010b)

. This study tested the influence of the test substance which is poorly soluble and its degradation products on growth inhibition under closed conditions. Briefly two stock solutions were first prepared at loading rates of 1 mg/L and 100 mg/L with test media. Stock solutions were stirred for 72h (the time believed from preliminary studies to be the optimal time for maximising the parent compound concentration in aqueous solution) and then left in a separation funnel for phase separation. Middle layers of the stock solutions were then split into two batches: one batch for direct use and a second aged for 7 days, in order to allow hydrolysis products of the substance to be present should it be likely to degrade abiotically. After 72h of exposure, mean measured concentrations in the first test were respectively 0.089 mg/L for the stock solution at a loading rate of 1 mg/L and 1.0 mg/L for the stock solution at a loading rate of 100 mg/L. Both of these concentrations are known to be well over the aqueous solubility limit of the substance. No effects were observed in either of the groups (parent or potential degradation products tested. It was concluded that neither the substance nor potential degradation products had any effects up to the limit of solubility.