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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
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
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
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:
The concentrations of Dimethyl adipate in the working stock (nominal 100 mg Dimethyl adipate /L), blank control, and test concentration (nominal 6.25, 12.5, 25, and 50 mg Dimethyl adipate/L) solutions were verified at test initiation (day 0). An aliquot from each of the blank control and test concentration solutions was taken prior to the addition of Selenastrum capricornutum. The pH was determined for each of the primary stock, blank control, and test concentration solutions.

The concentrations of Dimethyl adipate in the blank control, test concentration (nominal 6.25, 12.5, 25, 50, and 100 mg Dimethyl adipate /L), and abiotic control (nominal 100 mg/L Dimethyl adiapte /L) solutions were verified at test termination (day 3). These samples were prepared by pooling all 3 replicates for each of the control and test concentration solutions and taking an aliquot from each of the pooled samples. The pH was determined for each of these day 3 samples.

The concentrations of Dimethyl adipate in each of the test solutions were determined by high performance liquid chromatography (HPLC).
Vehicle:
no
Details on test solutions:
Prior to the initiation of the definitive test, method development was conducted to determine the appropriate concentrations to be used in the definitive test.

A working stock solution was prepared by dissolving the test substance in 1000 mL filter sterilized AAP nutrient medium for a nominal concentration of 100 mg Dimethyl adipate /L (= 100 ppm). Aliquots of the filter-sterilized AAP nutrient medium were used for the blank (normal culture medium) control solution. Test solutions were prepared using aliquots of the nominal 100 mg/L working stock solution and diluting with filter-sterilized AAP nutrient medium to make nominal concentrations of 6.25, 12.5, 25, and 50 mg DMA/L. Aliquots of the nominal 100 mg/L working stock solution were used for the 100 mg DMA/L test concentration solution and the abiotic (stability) control solution.

For each definitive control and test solution, 3 aliquots (50 mL each) were placed in separate sterilized 250 mL Erlenmeyer flasks fitted with sterilized foam stoppers. Each flask, excluding the abiotic control, was randomly assigned a number to eliminate bias while counting. To achieve the desired nominal concentration of approximately 10,000 Selenastrum capricornutum cells/mL at test initiation, an approximate 0.625 mL (= 625 μL) aliquot of algal inoculum from a logarithmically growing stock culture was aseptically transferred to each flask, except the abiotic control.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The original culture source was the Department of Botany - Culture Collection of Algae – The University of Texas at Austin - Austin, Texas 78713-7640.

The culture method for Selenastrum capricornutum was based on published literature. Prior to the study, Selenastrum capricornutum cultures were maintained under the same environmental conditions used in the study. The organisms were cultured in sterilized 250 mL Erlenmeyer flasks containing approximately 50 mL of filtered (= filter-sterilized) AAP nutrient medium and were aseptically transferred to fresh medium every 3 to 7 days. The flasks were fitted with foam stoppers to permit gas exchange.

AAP nutrient medium was prepared by adding 1 mL of each of the 6 macronutrient stock solutions and 1 mL of the micronutrient stock solution to approximately 800 mL of Milli-Q (deionized) water, with mixing after each addition. The volume of the medium was brought to 1 liter with additional Milli-Q water. Appropriate proportions (1 mL of each stock solution for each 1 liter of medium) were used to prepare the larger volumes of the medium required for the definitive and recovery tests.

The medium pH was adjusted to 7.51 with 0.1N sodium hydroxide. For both the definitive and recovery tests, the medium was filter-sterilized using Corning pre-sterilized filtration systems each with a 0.22 μm cellulose acetate filter. The containers with the resulting filter-sterilized AAP nutrient medium for use in the definitive and recovery tests were stored in the refrigerator in the dark at approximately 4°C and acclimated to room temperature prior to use. Any remaining unused medium was properly stored.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
None of the definitive test concentrations exhibited a 50% or greater growth inhibition based on healthy cell count relative to the blank control. Therefore, a recovery test was not performed.
Hardness:
not applicable
Test temperature:
24.6°C
pH:
For the definitive test, the pH measurements of the test solutions ranged from 7.32 to 7.98 at the 0-hour and from 7.47 to 8.41 at the 72-hour interval
Dissolved oxygen:
not applicable
Salinity:
not applicable
Nominal and measured concentrations:
day 0 - nominal concentrations - 0, 6.25, 12.5, 25 and 50 mg/l,
day 0 - measured concentrations - 0, 5.5, 12, 25 and 49 mg/l
Details on test conditions:
The control and test flasks were placed in a chamber (Hotpack model 06084) and air temperature in the chamber was recorded continuously with a continuous temperature recorder (Dickson Model SL445C7). The algae were incubated for 72 hours without test medium renewal for the definitive test. Illumination was supplied by cool-white fluorescent tubes.

Selenastrum capricornutum growth measurement was determined by visually counting the number of cells taken from an approximate 0.2 mL sample from each flask at approximately 24, 48, and 72 hours from the definitive test initiation. The counts were conducted using a hemacytometer and a compound microscope. An aliquot of each sample was loaded into the hemacytometer and 16 grids were selected. All cells located in the 16 grids were counted and recorded as healthy or unhealthy. The total number of cells counted was multiplied by 10,000 to determine the number of cells per milliliter. Cells outside these 16 grids were not counted nor included in the total number. Observations and counts of healthy and unhealthy cells (e.g., deformed, scenescent, stunted) were recorded in the study records. Counts were made at approximately the same time each day.
Reference substance (positive control):
not required
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: both growth rate and cell number
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
12.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: both growth rate and cell number
Details on results:
The organisms were exposed for 72 hours without test medium renewal. The effects were expressed in terms of percent inhibition in growth based on healthy cell count, area under the growth curve, and growth rate relative to the blank control for the 72-hour interval of the test.
The 72-hour results are as follows:
Healthy Cell Count - EC50 > 100 mg Dimethyl adipate/L, NOEC 12.5 mg Dimethyl adipate/L,
Area Under the Growth Curve - EC50 > 100 mg Dimethyl adiapte/L, NOEC 50 mg Dimethyl adiapte/L,
Growth Rate - EC50 > 100 mg Dimethyl adipate/L, NOEC 12.5 mg Dimethyl adiapte/L
The reductions in healthy cell count, area under the growth curve, and growth rate indicate a dose-dependent response with increasing concentrations of the test substance
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
Standard statistical test procedures were employed.

None

Validity criteria fulfilled:
yes
Conclusions:
The reductions in healthy cell count, area under the growth curve, and growth for Selenastrum capricornutum at 72 hours (3 days) indicate a dose-dependent response for increasing concentrations of the test substance, DMA. The most sensitive parameters were healthy cell count and growth rate each with an EC50 of > 100 mg/L and a NOEC of 12.5 mg/L..
Executive summary:

A study was conducted to determine the effect of Dimethyl Adipate (DMA) on the growth and growth rate of the green alga Selenastrum capricornutum.a The algae were exposed to nominal concentrations of 6.25, 12.5, 25, 50, and 100 mg DMA/liter of nutrient medium (ppm).

For the definitive (dose-response) test, the organisms were exposed for 72 hours (3 days) without test medium renewal. The effect was expressed as percent inhibition in growth based on healthy cell count (cell density), area under the growth curve, and growth rate relative to the blank (normal culture medium) control for the 72-hour (day 3) interval of the test.

The 72-hour results are as follows:

Healthy Cell Count - EC50 > 100 mg Dimethyl adipate/L, NOEC 12.5 mg Dimethyl adipate/L,

Area Under the Growth Curve - EC50 > 100 mg Dimethyl adiapte/L, NOEC 50 mg Dimethyl adiapte/L,

Growth Rate - EC50 > 100 mg Dimethyl adipate/L, NOEC 12.5 mg Dimethyl adiapte/L

The reductions in healthy cell count, area under the growth curve, and growth rate indicate a dose-dependent response with increasing concentrations of the test substance.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
30-Apr-2010 to 22-May-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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.
Reason / purpose:
reference to same study
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
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 761/2009
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 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.

All samples were stored deep-frozen (at about -20 °C) immediately after sampling until analysis.

The concentrations of the test item were determined in the duplicate test medium samples from the nominal test concentrations of 46 and 100 mg/L. The samples from the nominal test concentrations of 4.6 to 22 mg/L 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.
Vehicle:
no
Details on test solutions:
The test medium of the highest nominal concentration of 100 mg/L was prepared by dissolving 40.39 mg of the test item completely in 400 mL of test water using ultrasonic treatment for 15 minutes and intense stirring for 10 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 four days before the start of the exposure. The algae were cultivated under the test conditions. The inoculum culture was diluted threefold one day before the start of the test to ensure that the algae were in the exponential growth phase when used to inoculate the test solutions.

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 6.8 and 8.4 for the additional not pH adjusted control replicates. At the end of the test, pH values of 7.5 to 9.2 were measured .
Dissolved oxygen:
not applicable
Salinity:
according to OECD test guideline
Nominal and measured concentrations:
The nominal concentrations of the test item of 4.6, 10, 22, 46 and 100 mg/L were tested in parallel with a control.

The measured concentrations of the test item in the test media of the nominal test concentrations of 46 and 100 mg/L were 99 and 100% of the nominal values, respectively, at the start of the test. During the test period of 72 hours, a decrease of test item concentration in the test media occurred. At the end of the test, 62 to 72% of the nominal values were found.
Details on test conditions:
Reconstituted test water prepared according to the test guidelines was used for algal cultivation and testing. For further information on the test water please see section- any other information on materials and methods incl. 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 7600 Lux (range: 6990 to 8310 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.

The following nominal concentrations of the test item were tested: 4.6, 10, 22, 46 and 100 mg/L. Additionally, a control was tested in parallel (test water without test item).

In consultation with the Sponsor, the pH of the test water was lowered to 6.8 in order to maintain the test item concentration in the test media as constant as possible.

The test design included three replicates per test concentration and six replicates of the control for which the pH was adjusted to 6.8. Three additional control replicates were prepared with test medium for which the pH was not lowered (pH: 8.4).

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.


Reference substance (positive control):
yes
Remarks:
twice a year
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
36 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.:
85 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
and yield
Details on results:
The measured concentrations of the test item in the test media of the nominal test concentrations of 46 and 100 mg/L were 99 and 100% of the nominal values, respectively, at the start of the test. During the test period of 72 hours, a decrease of test item concentration in the test media occurred. At the end of the test, 62 to 72% of the nominal values were found. The mean measured test concentrations (calculated as the geometric means of the concentrations measured at the start and the end of the test) were between 78 and 85% of the nominal values.

For further details on biological results please see section- any other information on results incl. tables.

The algal growth (biomass) in the control without pH adjustment (pH: 8.4) was not statistically significantly different from the control with pH adjustment after 72 hours of test duration (according to Student-t test, alpha = 0.05, two-sided). Thus, the pH adjustment of the test water in order to keep the test item concentrations as constant as possible had no (statistically significant) effect on the growth of the algae.

The test item had a statistically significant inhibitory effect on the growth of the algae (average growth rate and yield) after the test period of 72 hours at the mean measured concentration of 85 mg/L (results of Williams tests, one-sided smaller, alpha= 0.05, Table 2 and Table 3). Thus, this concentration was determined to be the 72 hour LOEC.

The 72-hour NOEC was determined to be 36 mg/L, since up to and including this mean measured test concentration, the growth rate and yield of the algae after 72 hours were not statistically significantly lower than in the control.

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.

In the control the biomass increased by a factor of 204 over 72 hours . 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) during 72 hours was 7.6%. 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 0.8%. 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 6.8 and 8.4 for the additional not pH adjusted control replicates. At the end of the test, pH values of 7.5 to 9.2 were measured. The water temperature during the test was maintained at 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 to 1.7 mg/L).
Reported statistics and error estimates:
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 and EC20 values for the inhibition of average growth rate and yield and their 95% confidence intervals were calculated as far as possible by Probit Analysis.

The 72-hour EC50 values of the test item could not be calculated due to the low 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 concentrations were compared to the control values by Williams tests and Welch-t tests .

Please see attached "Figures 1 to 3" for:

Figure 1 Growth Curves of the Algae over the Test Duration of 72 Hours

Figure 2 Concentration-Effect Relationship of Average Growth Rates after 72 Hours

Figure 3 Concentration-Effect Relationship of Yield after 72 Hours

The biological results were related to the mean measured test item concentrations:

 

Nominal test concentration

Mean measured concentration of the test item
(geometric mean)

(mg/L)

(mg/L)

(% of nominal)

4.6

---

---

10

---

---

22

---

---

46

36

78

100

85

85

 

---:      not analyzed,

The biological results can be summarized as follows (on the basis of mean measured concentrations of the test item):

 

Parameter

Growth rate

Yield

(0-72 h)

 

 

EC10  (mg/L)

>85

73

95% confidence interval

n.d.

50 - 86

EC20  (mg/L)

>85

>85

95% confidence interval

n.d.

n.d.

EC50  (mg/L)

>85

>85

95% confidence interval

n.d.

n.d.

NOEC (mg/L)

36

36

LOEC (mg/L)

85

85

 

n.d.  could not be determined

Table 1      Biomass of Algae

Nominal test item concentration (mg/L)

Mean measured concentration (mg/L)

Rep. no.

Biomass of algae*

24 hours

48 hours

72 hours

Control

Control

1

6.6

40.1

225.2

 

 

2

6.4

42.8

214.6

 

 

3

5.6

34.8

214.5

 

 

4

6.5

39.6

226.1

 

 

5

6.1

48.3

236.1

 

 

6

5.6

42.4

234.6

 

 

Mean

6.1

41.4

225.2

 

 

SD

0.5

4.4

9.3

Control without

Control without

1

4.4

32.2

212.4

pH adjustment

pH adjustment

2

6.1

36.1

239.6

 

 

3

6.0

44.0

257.2

 

 

Mean

5.5

37.4

236.4

 

 

SD

0.9

6.0

22.6

4.6

---

1

6.1

40.5

222.6

 

 

2

6.4

40.2

246.9

 

 

3

5.8

43.0

231.9

 

 

Mean

6.1

41.2

233.8

 

 

SD

0.3

1.5

12.3

10

---

1

6.5

41.7

211.5

 

 

2

6.1

38.6

243.4

 

 

3

5.9

39.2

217.0

 

 

Mean

6.1

39.8

223.9

 

 

SD

0.3

1.6

17.0

22

---

1

6.5

38.9

223.7

 

 

2

5.0

28.8

215.6

 

 

3

6.1

44.1

252.7

 

 

Mean

5.9

37.2

230.7

 

 

SD

0.8

7.8

19.5

46

36

1

6.2

40.6

214.4

 

 

2

6.1

37.4

219.3

 

 

3

5.7

40.1

229.2

 

 

Mean

6.0

39.4

221.0

 

 

SD

0.3

1.7

7.5

100

85

1

5.2

37.9

191.7

 

 

2

5.9

36.0

198.4

 

 

3

5.2

35.6

198.8

 

 

Mean

5.5

36.5

196.3

 

 

SD

0.4

1.2

4.0

 

SD:  Standard deviation

*:     The biomass was determined by fluorescence measurement (at least duplicate measurements per replicate) and is given as relative fluorescence units (x 103). At the start of the test, the initial cell density was 10000 algal cells/mL, corresponding to 1.10 x 103relative fluorescence units.

--:    not analyzed

Table 2      Average Growth Rates (µ)

Nominal
test item concentration

Mean measured concentration

Average growth rate µ (day-1) and inhibition of µ (Ir)

0-24 h

0-48 h

0-72 h

(mg/L)

(mg/L)

µ

Ir(%)

µ

Ir(%)

µ

Ir(%)

Control

---

1.71

0.0

1.81

0.0

1.77

0.0

4.6

---

1.70

0.4

1.81

0.0

1.78

-0.7

10

---

1.72

-0.3

1.79

0.9

1.77

0.1

22

---

1.67

2.3

1.75

3.2

1.78

-0.4

46

36

1.69

1.2

1.79

1.2

1.77

0.3

100

85

1.60*

6.6

1.75

3.3

1.73*

2.6

 

*:          mean value statistically significantly lower than in thecontrol
(according to Williams-test, one-sided smaller,
a= 0.05)

---:        not analyzed

 

 

Table 3      Yield (Y)

Nominal
test item concentration

Mean measured concentration

Yield Y (x 103) and inhibition of Y (Iy)

0-24 h

0-48 h

0-72 h

(mg/L)

(mg/L)

Y

Iy(%)

Y

Iy(%)

Y

Iy(%)

Control

---

5.0

0.0

40.2

0.0

224.1

0.0

4.6

---

5.0

0.9

40.1

0.3

232.7

-3.8

10

---

5.0

-0.4

38.7

3.8

222.8

0.6

22

---

4.8

4.3

36.1

10.2

229.6

-2.5

46

36

4.9

2.7

38.3

4.9

219.9

1.9

100

85

4.4*

13.1

35.4

12.1

195.2*

12.9

 

*:          mean value statistically significantly lower than in thecontrol
(according to Williams-test, one-sided smaller,
a= 0.05)

---:        not analyzed

 

Table 4      Section-by-Section Growth Rates

Nominal
test item concentration

Mean measured concentration

Section-by-section growth rates (day-1)
and inhibition of the growth rates (Ir)

0-24 h

24-48 h

48-72 h

(mg/L)

(mg/L)

µ

Ir(%)

µ

Ir(%)

µ

Ir(%)

Control

---

1.71

0.0

1.91

0.0

1.70

0.0

4.6

---

1.70

0.4

1.91

-0.4

1.73

-2.1

10

---

1.72

-0.3

1.87

2.0

1.73

-1.6

22

---

1.67

2.3

1.83

4.0

1.84

-8.1

46

36

1.69

1.2

1.88

1.3

1.73

-1.6

100

85

1.60

6.6

1.90

0.4

1.68

0.9

 

---:          not analyzed

 

 

Table 5      pH Values in theTreatments

Nominal
test item concentration

pH values

(mg/L)

Start

End

Control without pH adjustment

8.4

9.2

Control

6.8

8.2

4.6

6.8

8.2

10

6.8

8.2

22

6.8

8.0

46

6.8

8.0

100

6.8

7.5

Validity criteria fulfilled:
yes
Conclusions:
The test item had a statistically significant inhibitory effect on the growth of the algae (average growth rate and yield) after the test period of 72 hours at the mean measured concentration of 85 mg/L (results of Williams tests, one-sided smaller, α = 0.05, Table 2 and Table 3). Thus, this concentration was determined to be the 72 hour LOEC.

The 72-hour NOEC was determined to be 36 mg/L, since up to and including this mean measured test concentration, the growth rate and yield of the algae after 72 hours were not statistically significantly lower than in the control.
Executive summary:

The influence of the test item 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 761/2009, C.3.

This study has been performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice, adopted 2005 [SR 813.112.1]. This ordinance is based on the OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted November 26th, 1997 by decision of the OECD Council [C(97) 186/Final]. Theses principles are compatible with Good Laboratory Practice regulations specified by regulatory authorities throughout the European Community, the EPA and FDS and MHLW, MAFF and METI.

 

The nominal concentrations of the test item of 4.6, 10, 22, 46 and 100 mg/L were tested in parallel with a control.

 

The measured concentrations of the test item in the test media of the nominal test concentrations of 46 and 100 mg/L were 99 and 100% of the nominal values, respectively, at the start of the test. During the test period of 72 hours, a decrease of test item concentration in the test media occurred. At the end of the test, 62 to 72% of the nominal values were found.

 

The mean measured concentrations (calculated as the geometric means of the concentrations measured at the start and the end of the test) in the test concentrations of 46 and 100 mg/L were 36 and 85 mg/L, respectively. The nominal test concentrations of 4.6 to 22 mg/L were not analyzed, since these concentrations were below the NOEC determined in this test.

 

The biological results (based on mean measured test item concentrations) were as follows:

 

Parameter

Growth rate

Yield

(0-72 h)

 

 

EC10  (mg/L)

>85

73

95% confidence interval

n.d.

50 - 86

EC20  (mg/L)

>85

>85

95% confidence interval

n.d.

n.d.

EC50  (mg/L)

>85

>85

95% confidence interval

n.d.

n.d.

NOEC (mg/L)

36

36

LOEC (mg/L)

85

85

 

n.d.   could not be determined

All the validity criteria were fulfilled.

Description of key information

The 72-hour EC50 (growth rate and yield) of DBE is estimated to be greater than 85 mg/l in the OECD 201 test.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
36 mg/L

Additional information

Justification for Read Across

Attached at section 13 is a category justification that explains the rationale for using data on a mixture of Dimethyl Esters (DBE), and Dimethyl Glutarate, to supplement the available data on Dimethyl Adipate.

Two reliable key studies are available for this endpoint. In one study (Weber B., 2010) the influence of the DBE on the growth of the freshwater green algal speciesPseudokirchneriella 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 761/2009, C.3.

This study has been performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice, adopted 2005 [SR 813.112.1]. This ordinance is based on the OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted November 26th, 1997 by decision of the OECD Council [C(97) 186/Final]. Theses principles are compatible with Good Laboratory Practice regulations specified by regulatory authorities throughout the European Community, the EPA and FDS and MHLW, MAFF and METI.

 

The nominal concentrations of the test item of 4.6, 10, 22, 46 and 100 mg/L were tested in parallel with a control.

 

The measured concentrations of the test item in the test media of the nominal test concentrations of 46 and 100 mg/L were 99 and 100% of the nominal values, respectively, at the start of the test. During the test period of 72 hours, a decrease of test item concentration in the test media occurred. At the end of the test, 62 to 72% of the nominal values were found.

 

The mean measured concentrations (calculated as the geometric means of the concentrations measured at the start and the end of the test) in the test concentrations of 46 and 100 mg/L were 36 and 85 mg/L, respectively. The nominal test concentrations of 4.6 to 22 mg/L were not analyzed, since these concentrations were below the NOEC determined in this test.

 

The biological results (based on mean measured test item concentrations) were as follows:

 

Parameter

Growth rate

Yield

(0-72 h)

 

 

EC10  (mg/L)

>85

73

95% confidence interval

n.d.

50 - 86

EC20  (mg/L)

>85

>85

95% confidence interval

n.d.

n.d.

EC50  (mg/L)

>85

>85

95% confidence interval

n.d.

n.d.

NOEC (mg/L)

36

36

LOEC (mg/L)

85

85

 

n.d.   could not be determined

All the validity criteria were fulfilled.

A second key study (Dupont, 2003) assessed the effect of dimethyl adipate on Selenastrum capricornutum. The reductions in healthy cell count, area under the growth curve, and growth for Selenastrum capricornutum at 72 hours (3 days) indicate a dose-dependent response for increasing concentrations of the test substance, DMA. The most sensitive parameters were healthy cell count and growth rate each with an EC50 of > 100 mg/L and a NOEC of 12.5 mg/L.