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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Adequacy of study:
other information
Qualifier:
according to
Guideline:
other: EWG 92/69
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Total exposure duration:
72 h
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1 mg/L
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.2 mg/L
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.27 mg/L
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.72 mg/L
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.19 mg/L
Basis for effect:
biomass
Executive summary:

The study measured EC50 and NOEC levels for green algae.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Conclusions:
Measured effect concentrations [mg/l]:

EC50 (72h): 1.72 (Scenedesmus subspicatus)
EC50 (48h): 3.50 (biomass) - 9.0 (growth rate) (Scenedesmus subspicatus)

Lowest observed NOEC: 0.2 (8d, Dunaliella parva)
0.8 (10d, Scenedesmus subspicatus)
Executive summary:

 Short-term toxicity of DBP to algae

 

No.

Species

Result(mg/l)

Method

Reference

1

Scenedesmus subspicatus

1.2 (72 hEC50)

92/69/EEC

Hüls(1995)

2

Scenedesmus subspicatus

3.5 (48 hEC50; biomass)

DIN38412/9

Kühn and Pattard (1990)

3

Scenedesmus subspicatus

9.0 (48 hEC50; growth rate)

DIN38412/9

Kühn and Pattard (1990)

4

Gymnodium breve

0.0034 -0.2(96hEC50)

Other

Wilson et al.(1978)

The test with the marine dinoflagellate Gymnodium breve showed a very poor reproducibility. In the first assay an EC50 of 0.0034 mg/l was established, whereas in the second, a value of 0.2 mg/l was found. It is doubtful whether such a large difference can be attributed to biological variation. This is supported by the fact that the variation between the replicates was much smaller in the tests with other phthalate esters in the same study. The BUA report (BUA, 1987) revealed some more, technical shortcomings of the Gymnodium test. For these reasons the test results will not be used for the derivation of the PNEC for the aquatic compartment. The NOEC values for both freshwater and marine algae are presented in following Table:

NOEC values of DBP for algae

 

No.

Species

Result(NOECinmg/l)

Method

Reference

1

Selenastrum capricornutum

2.8(7d)

Other

Melinand Egnéus (1983)

2

Selenastrum capricornutum

0.8(10d)

EGG-CMA-002

CMA (1984)

3

Skeletenoma costatum

0.6-0.7(?d)

Other(marine)

Medlin(1980)

4

Dunaliella parva

0.2(8d)

Other(marine)

Aceyetal.(1987)

5

Thalassiosira pseudomona

2.0(4d)

Other(marine)

Aceyetal.(1987)

6

Synecchococcus lividus

0.002(14dLOEC)

Other(marine)

Aceyetal.(1987)

It is necessary to discuss the 14-day LOEC of 0.002 mg/l for the blue-green algae Synecchococcus lividus in more detail. At DBP concentrations of 0.002 mg/l and higher, the number of monodispersed (i.e. non-aggregated) cells of the blue-green algae was found to be significantly decreased. This effect, however, can be fully attributed to a DBP induced shift from non- aggregated towards aggregated cells. At each concentration of DBP tested namely, the percentage of aggregated S. lividus was found to be increased: at 0.002 mg/l about 95% of the algae were in aggregated form as opposed to 22% in the control group. In addition, when counting the total number of S. lividus, i.e. both aggregated and non-aggregated organisms, a significant increase was found at all test concentrations. In conclusion it can be said that DBP caused a decrease only in the number of non-aggregated S. lividus. Nevertheless, very low concentrations of DBP seem to affect the growth behaviour of these blue-green algae. At present, however, the ecological significance of this effect is unknown and therefore the value will not be used for the PNEC derivation.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well documented and scientifically acceptable, according to German DIN guideline
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
other: DIN 38412/9
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Stock cultures: The green alga Scenedesmus subspicatus served as the test organism (strain number 8681 SAG), a strain which we have maintained over a number of decades in submerged culture. For this purpose, the required number of 100 ml Erlenmeyer flasks with metal caps containing 20 ml nutrient solution (see Table 1) was sterilized in a steam sterilizer for 30 min on 2 consecutive days. After cooling, the contents of each flask were inoculated with 2 ml cell suspension taken from a 10-day old stock culture. The inoculated flasks were placed on a white surface, protected from daylight and exposed to constant lighting from two parallel fluorescent Osram 40 W/30 tubes (distance from each tube 60 cm: irradiance E0, Sy = 24.9 W/m 2) at 24 +/-1°C and relative humidity of 50%. To maintain the test strain, fresh stock cultures were prepared at 10-day intervals.

Preliminary culture: The cultivation of the preliminary cultures was undertaken 3 days prior to the preparation of the test solution. For this purpose, 50 ml nutrient solution (for test cultures) (see Test Procedure) were filled into 300 ml Erlenmeyer flasks with metal caps and inoculated with Scenedesmus from 7-day-old stock cultures. The cell concentration in the preliminary culture flasks must amount to 10^4ml^-1 This was to ensure that the culture was still in a process of logarithmic growth after 72 h. Light and temperature conditions corresponded to those for the test preparation. The cell material of the preliminary cultures was used after 72 h to inoculate the dilution preparation after the cell concentration had been fixed at 10^5ml^-1.
Total exposure duration:
48 h
pH:
8.0 +/-0.3
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
1.4 mg/L
Remarks on result:
other: EbC10
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
2.6 mg/L
Remarks on result:
other: EµC10
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
3.5 mg/L
Remarks on result:
other: EbC50
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
9 mg/L
Remarks on result:
other: EµC50

The information on the pollutant effect of the substance was related to the nominal concentration and only in a few, clearly indicated cases, to the chemical quantification of the stock solution. The dilution quotient for the test preparations in all test series was 1 : 2.

Table 3 gives the results for the 43 substances which were tested according to a modified procedure of the Standard, i.e. in 250 ml bottles with groundglass stoppers and with an overall test time of 48 h. The following data were given for each substance:

EbC10 (0-48 h)

EbC50 (0-48 h)

EµC10 (0 -48 h)

EµC50 (0-48 h)

tested concentration range.

In the group of esters, three of the tested phthalates stood out. The longer the alkyl chains, the more toxic were the compounds. The substance concentrations for the 48 h-EbC10 in the case of dibutyl ester phthalate (1.4mg/l) and diallyl ester phthalate (l.9mg/l) were 6-5 times lower than for diethyl ester phthalate (11 mg/l).

Executive summary:

The study measured EC10 and EC50 values for green algae for 78 substances. Only those concerning DBP are documented here.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: well documented and scientifically acceptable, but lacking testing guidelines
Reason / purpose:
reference to other study
Principles of method if other than guideline:
No guideline specified, cf. "Any other information on material and methods" for details.
GLP compliance:
not specified
Test organisms (species):
other: Chlorella emersonii, Selenastrum capricornutum
Details on test organisms:
Pure stock cultures of Chlorella emersonii (CCAP strain 211/8 h), and Selenastrum capricornutum (CCAP strain 278/4) were maintained on proteose-peptone agar slopes (List of Strains 1976). Axenic cultures of Chloretla were grown in a turbidostat as described in Egneus and Blanck (1977). Axenic batch cultures of Selenasirum were grown in a 10% (v/v) Z-8 medium (Kotain 1972).
Test type:
static
Test temperature:
20 +/-1° C
Details on test conditions:
Exponentially growing cells from axenic cultures were used as inoculum for the test cultures. The algae were added to sterile flasks containing 100 ml sterile growth medium and DBP of varying concentrations. The initial population density corresponded to 0.1 µg chlorophyll /ml. The cultures were kept at a temperature of 20 ± 1°C and continuously illuminated with white light (General Electric F72P617 CW, USA) from above in a growth chamber. The light intensity was 20 W m^-2. Three culture flasks were used for each concentration of DBP. Growth of algae was determined by measuring changes in transmittance at 720 nm. Growth curve were constructed by plotting log2 A720nm + 10 against time (Sorokin 1973) and samples of 4 ml were taken every twelfth hour.
Duration:
7 d
Dose descriptor:
IC50
Effect conc.:
0 other: M
Basis for effect:
other: oxygen evolution

Exposure of Selenastrum to DBP in concentrations between 10 ^-5 and 10^-4 M lead to an inhibition of growth (Fig. 1). Concentrations higher than 10^-4 killed the algae. The growth during the first 24 h in 10^-4 M treated algae is due to cell division because of DBP-stress. Similar results were obtained with Chlorella emersonii. To investigate whether the effect of DBP on growth was due to an effect on photosynthesis, we studied the effect of DBP on CO2-dependent oxygen evolution from Chlorella (Fig. 2). The algae were incubated with DBP for 40 min before irradiation. The number of algae /ml was considerably higher in the photosynthesis experiments than in the growth experiments. DBP affected photosynthesis at concentrations higher than 10^-5 M, but the inhibition was weak, and at the highest concentration investigated oxygen evolution was only inhibited to about 50%.

Conclusions:
Di-n-butyl phthalate inhibits growth and photosynthesis of green algae (Chtorella emersonii CCAP strain 211/8 h anA Selenastrum capricornuium CCAP strain 278/4) at concentrations higher than 10^-5 M. The IC50 value for CO2-dependent oxygen evolution in algae was 3 x 10^-4 M. Di-n-butyl phthalate could thus be a pollutant which affects growth and photosynthesis of plants.
Executive summary:

This is part of a srudy conducted on algae, barley and spinat. Only the effects on algae are documented here. Those on barley and spinach can be found under section 6.3.3

Description of key information

Measured effect concentrations [mg/l]:
EC50 (72h): 1.72 (Scenedesmus subspicatus)
EC50 (48h): 3.50 (biomass) - 9.0 (growth rate) (Scenedesmus subspicatus)
EC50 (96h): 30.2 (Scenedesmus. obliquus)
Lowest observed NOEC: 0.2 (8d, Dunaliella parva)
0.8 (10d, Scenedesmus subspicatus)

Key value for chemical safety assessment

EC50 for freshwater algae:
1.72 mg/L
EC10 or NOEC for freshwater algae:
0.2 mg/L

Additional information

Freshwater algae Scenedesmus obliquus treated with DBP showed an EC50 (96h) of 30.2 mg/L. Testing on natural algae showed the inhibition percentage of DBP was lower with same DBP concentrations and exposure time. This suggests that different algal species had varying reactions to DBP. For the chemical safety assessment the most sensitive endpoint (Scenedesmus subspicatus) was chosen.